Nucleotide sequences encoding ramosa3 and sister of ramosa3 and methods of use for same

ABSTRACT

The invention relates to the isolation and characterization of a maize gene, RAMOSA3 (RA3), responsible for meristem development and inflorescence development including branching. The gene, gene product, and regulatory regions may be used to manipulate branching, meristem growth, inflorescence development and arrangement, and ultimately to improve yield of plants. The invention includes the gene and protein product as well as the use of the same for temporal and spatial expression in transgenic plants to alter plant morphology and affect yield in plants. The invention also includes the gene and protein product for SISTER OF RAMOSA3 (SRA).

This application claims the benefit of U.S. Provisional Application No.60/642,273, filed Jan. 7, 2005, and U.S. Provisional Application No.60/739,857, filed Nov. 23, 2005, each of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The field of invention relates generally to plant molecular biology, andmore specifically, to protein and nucleotide sequences and genetictechniques using the same to modify plant architecture and to increaseyield and health of plants.

BACKGROUND OF THE INVENTION

Plant architecture is central to yield, for example in orchestrating thegreen revolution through reducing plant height [Peng, J. et al., Nature,1999. 400(6741): p. 256-611]. Similarly, inflorescence morphology is amajor yield factor in many crops, and is determined by the activities ofshoot meristems. Variations in branching patterns lead to diversity inarchitectures, and have been studied at the genetic, physiological andmolecular levels [Sussex, I. M. and Kerk, N. M., Curr Opin Plant Biol,2001. 4(1): p. 33-7; Ward, S. P. and Leyser, O., Curr Opin Plant Biol,2004. 7(1): p. 73-8].

Factors that Control Shoot Branching

The shoot apical meristem (SAM) is active throughout the plantlifecycle, and produces leaves and axillary meristems, typicallyinitiated in each leaf axil. Patterns of axillary meristemactivity—where they are produced, whether they undergo dormancy orgrowth and how much they grow—contribute to overall shoot architecture.Apical dominance is a major factor in regulating development of axillarymeristems, and is regulated by genetic and hormonal factors.

Development of axillary meristems can be described in two phases,initiation and growth.

Genes like REVOLUTA (REV) and LATERAL SUPPRESSOR (LAS) in Arabidopsisare involved in initiation. rev mutants often fail to produce axillarymeristems during both vegetative and reproductive development. REVencodes a homeodomain/leucine zipper transcription factor and isexpressed in very early axillary meristems. On the other hand, in lasmutants, defects in formation of axillary meristems occur only duringvegetative development. LAS is however expressed in leaf axils duringboth vegetative and reproductive development. LAS also encodes atranscriptional factor, a member of the GRAS family. Expression patternssuggest that LAS acts upstream of REV in axillary meristem development.

The maize teosinte branched1 (tb1) locus is another well-characterizedregulator of axillary meristem growth. The tb1 mutant phenotyperesembles the maize ancestor, teosinte, in axillary meristem outgrowth.QTL analysis, gene structure and expression analyses indicate that TB1is one of the major genes involved in the important change of plantarchitecture involved in the development of maize as a crop. The riceTB1 ortholog appears to act downstream of the rice LAS ortholog,monoculm1.

The regulation of axillary meristem outgrowth is complex, and severalgenes, as well as the hormones, auxin, cytokinin and abscisic acid (ABA)have been implicated. Auxin is traditionally thought of as an inhibitorof axillary meristem growth, though recent evidence suggests that italso functions during axillary meristem initiation. The importance ofauxin for growth is suggested by the fact that auxin resistant1 (axr1)mutants, which have reduced responses to auxin, are highly branched.However, the concentrations of auxin in axillary meristems afterdecapitation are sometimes higher than before, and auxin appears to actnon-autonomously, in the xylem and interfascicular schlerenchyma, tosuppress branching. This suggests the existence of mobile secondmessengers that transmit the auxin signal.

One candidate is for such a signal is a cytokinin. Cytokinins canpromote the growth of axillary meristems following direct application,and auxin can down-regulate cytokinin biosynthesis. Further evidence ofa role for cytokinin in axillary meristem growth comes from analysis ofthe Arabidopsis hoc and supershoot mutants, which have higher endogenouscytokinin levels and an extremely branched phenotype. Physiological andgenetic analyses suggest that the hormone abscisic acid may alsoregulate the growth of axillary meristems.

Genetic analysis is also beginning to identify novel branching signals.ramosus (rms) mutants in pea and more axillary growth (max) mutants inArabidopsis have a bushy phenotype and auxin resistant bud growth.Grafting experiments, hormone measurements and responses of thesemutants to auxin strongly suggested the existence of a novel mobilebranch-inhibition signal that functions downstream of auxin. Recentcloning of MAX3 and MAX4 has started to provide some clues as to thenature of this signal. Both genes encode proteins related todioxygenases, and the hypothesis is that the signal is derived bycleavage of a carotenoid. Analysis of other rms and max mutants hasrevealed other genes that are not rescued by grafting, and may functionin MAX/RMS-dependent signal perception. The fact that MAX2 encodes anF-box protein may match the hypothesis, since many such proteins areinvolved in hormone regulation.

Factors that Control Inflorescence Branching

Internal and environmental stimuli promote the transition fromvegetative SAM to inflorescence meristem (IM), which initiates theinflorescence structures. In grasses, inflorescence development ischaracterized by the formation of short branches called spikelets. Maizeforms two distinct types of inflorescence; the terminal tassel has longbranches and develops the male flowers, and the axillary ears have aprominent axis lacking long branches, and develop female flowers. The IMinitiates files of spikelet pair meristems (SPMs), and each SPM in turnproduces two spikelet meristems (SMs), which initiates two floralmeristems (FMs). In the tassel, the IM also initiates several branchmeristems (BMs), which are responsible for the long branches at the baseof the tassel, followed by SPMs, SMs and FMs as in the ear.

Inflorescence architecture in different species is highly variable, forexample in contrast to maize, rice has only one kind of bisexualinflorescence formed from apical and axillary meristems. Each IMproduces primary branches in a spiral phyllotaxy, and these makesecondary branches and SMs in a biased distichous phyllotaxy. Bothprimary and secondary branch meristems (BMs) in rice correspond to SPMsin maize inflorescences, from the viewpoint that they initiate SMs. Therice IM degenerates after making primary BMs, and the internodes of bothprimary and secondary branches elongate to form a panicle architecture,which looks very different from that of maize, though their structuralcomponents are essentially the same.

Several genes that regulate maize and rice inflorescence branching andmorphology have been identified. BARREN INFLORESCENCE2 (BIF2) and BARRENSTALK1 (BA1) in maize, and LAX PANICLE (LAX) in rice control theinitiation of axillary IMs. bif2 tassels make fewer or no branches andspikelets, and the ears have fewer or no spikelets. Since the BMs, SPMs,SMs and FMs in weak bif2 mutants are all defective, it appears that BIF2is required for initiation and maintenance of all types of axillarymeristems. ba1 mutants lack vegetative axillary branches and ears, andhave unbranched tassels lacking spikelets. In rice lax mutants, thenumber of primary branches and spikelets is also strongly reduced. Thesephenotypes indicate that both BA1 and LAX are required to initiateinflorescence axillary meristems. They encode orthologous basichelix-loop-helix transcription factors, and are expressed at theboundaries between pre-existing and newly initiated axillary meristems.These localized expression patterns support the functions of LAX and BA1in the production of axillary meristems, and suggest that the genefunctions are strongly conserved between rice and maize. Zea floricaulaI leafy (zfI) mutants in maize also have less tassel branches and fewerear row numbers in addition to defects during the inflorescencetransition.

Once axillary meristems are initiated, they acquire new identities.RAMOSA1 (RA1) in maize is required in the tassel and ear for thetransition from SPM to SM identity, and ra1 mutants have highly branchedinflorescences [Postlethwait, S. N. and Nelson, O. E., Characterizationof development in maize through the use of mutants. I. The polytypic(Pt) and ramosa-1 (ra1) mutants. Am. J. Bot., 1964. 51: p. 238-243;Vollbrecht et al. Nature 436:1119-1126].

Ramosa2 (ra2) mutants have a similar phenotype to ra1 except that in ra2the pedicellate spikelet is converted to a branch. Nickerson, N. H. etal., Tassel modifications in Zea mays. Ann. MO Bot. Gard., 42, 195-211(1955); Hayes, H., Recent linkage studies in maize. IV. Ramosa ear-2(ra2). Genetics, 1939. 24: p. 61.

Ramosa3 (ra3) is a classical mutant of maize, first described in 1954[Perry, H. S., Unpublished, http://www.maizegdb.org/.1954] (see also,Table 1 of Veit et al. Plant Cell 5:1205-1215 (1993)), but it has notbeen characterized in detail. Only the mature inflorescence phenotypehas been reported.

In tassel seed4 (ts4) mutants most SPMs in the tassel and those on thedistal part of the ear reiterate SPMs, therefore TS4 is required for SMidentity. FRIZZY PANICLE (FZP) in rice and BRANCHED SILKLESS1 (BD1) inmaize regulate meristem identity at the transition from SMs to FMs. fzpand bd1 mutants produce branching structures without making flowers, sothese genes are required to regulate the determinacy of SMs and/or toestablish the identity of FMs. FZP and BD1 encode orthologs in theethylene-responsive element-binding factor class of transcriptionfactors. They are expressed in analogous patterns at the junction of SMsand rudimentary glumes in rice, and SMs and inner/outer glumes in maize.Therefore some genes that regulate inflorescence architecture in riceand maize are strongly conserved in function and expression pattern.

Other genes regulating SM determinacy include the maize REVERSED GERMORIENTATION1 (RGO1), INDETERMINATE SPIKELET1 (IDS1), INDETERMINATEFLORAL APEX1 (IFA1) and TASSEL SEED6 (T56) genes. In these mutants, theSMs become more indeterminate, and produce extra flowers. The degree ofSPM and SM determinacy is one of the characteristic variables in grassinflorescence architecture, and differs significantly between rice andmaize, as described above, and in other grasses, for example, in wheat,the SMs are indeterminate.

Genetic and molecular analyses in model species have contributed tounderstanding the mechanisms of inflorescence branching in the grasses.Most of the genes that have been isolated encode putative transcriptionfactors, suggesting they regulate the transcription of downstreamtargets.

Trehalose Biology and Signaling

Trehalose is a disaccharide composed of two glucose units. It is highlyresistant to heat and pH and has a strong stabilizing effect onproteins. In contrast to sucrose, which is present only in the plantkingdom and some photosynthetic prokaryotes, trehalose is present in allkingdoms and plays a role in carbohydrate storage and stress protectionin microbes and invertebrates. See, e.g., Goddijn, O. J. and van Dun,K., Trehalose metabolism in plants. Trends Plant Sci, 1999. 4(8): p.315-319; Elbein, A. D., The metabolism of a,a-trehalose. Adv. Carbohydr.Chem. Biochem., 1974. 30(227-56); Crowe, J. H., Hoekstra, F. A., andCrowe, L. M., Anhydrobiosis. Annu Rev Physiol, 1992. 54: p. 579-99;Strom, A. R. and Kaasen, I., Trehalose metabolism in Escherichia coli:stress protection and stress regulation of gene expression. MolMicrobiol, 1993. 8(2): p. 205-10; and Paiva, C. L. and Panek, A. D.,Biotechnological applications of the disaccharide trehalose. BiotechnolAnnu Rev, 1996. 2: p. 293-314.

Until recently trehalose was not thought to be present in vascularplants, with the exception of desiccation-tolerant plants (reviewed inMuller, J., Boller, T., and Wiemken, A., Trehalose and trehalase inplants: recent developments. Plant Sci., 1995. 112: p. 1-9).

However, interest in the application of engineering trehalose metabolismto produce drought tolerant crops led to the discovery of trehalosebiosynthetic genes in plants. See, e.g., Holmstrom, K. O., Mantyla, E.,Welin, B., Mandal, A., and Palva, E. T., Drought tolerance in tobacco.Nature, 1996. 379: p. 683-684; Romero, C., Belles, J. M., Vaya, J. L.,Serrano, R., and Cilianez-Macia, A., Expression of the yeasttrehalose-6-phosphate synthase gene in transgenic tobacco plants:pleiotropic phenotypes include drought tolerance. Planta, 1997. 201: p.293-297; and Garg, A. K., Kim, J. K., Owens, T. G., Ranwala, A. P.,Choi, Y. D., Kochian, L. V., and Wu, R. J., Trehalose accumulation inrice plants confers high tolerance levels to different abiotic stresses.Proc Natl Acad Sci USA, 2002. 99(25): p. 15898-903. See also, e.g.,Goddijn, O. J., Verwoerd, T. C., Voogd, E., Krutwagen, R. W., de Graaf,P. T., van Dun, K., Poels, J., Ponstein, A. S., Damm, B., and Pen, J.,Inhibition of trehalase activity enhances trehalose accumulation intransgenic plants. Plant Physiol, 1997. 113(1): p. 181-90; Muller, J.,Aeschbacher, R. A., Wingler, A., Boller, T., and Wiemken, A., Trehaloseand trehalase in Arabidopsis. Plant Physiol, 2001. 125(2): p. 1086-93;Vogel, G., Fiehn, O., Jean-Richard-dit-Bressel, L., Boller, T., Wiemken,A., Aeschbacher, R. A., and Wingler, A., Trehalose metabolism inArabidopsis: occurrence of trehalose and molecular cloning andcharacterization of trehalose-6-phosphate synthase homologues. J ExpBot, 2001. 52(362): p. 1817-26; and Leyman, B., Van Dijck, P., andThevelein, J. M., An unexpected plethora of trehalose biosynthesis genesin Arabidopsis thaliana. Trends Plant Sci, 2001. 6(11): p. 510-3.

N-terminal deletion of the Selaginella lepidophylla (a “resurrectionplant”) or Arabidopsis thaliana trehalose-6-phosphate synthase (TPS1)results in a dramatic increase in TPS activity (Van Dijck et al., 2002,Biochem. J. 366:63-71). This indicates a high potential trehalosesynthesis capacity in plants in spite of the near universal absence oftrehalose.

The biosynthesis of trehalose occurs in 2 steps, analogous to that ofsucrose. Trehalose-6-phosphate (T6P) is first formed from UDP-glucoseand glucose-6-phosphate by trehalose-6-phosphate synthase (TPS). Next,T6P is converted to trehalose by trehalose-6-phosphate phosphatase(TPP).

Trehalose metabolism has been analyzed in detail in Saccharomycescerevisiae and Escherichia coli. In S. cerevisiae, TPS1 is responsiblefor TPS activity, and TPS2 for TPP activity, and they function in acomplex together with regulatory subunits encoded by TPS3 and TSL1[Londesborough, J. and Vuorio, O., Trehalose-6-phosphatesynthase/phosphatase complex from bakers' yeast: purification of aproteolytically activated form. J Gen Microbiol, 1991. 137 (Pt 2): p.323-30; Bell, W., Sun, W., Hohmann, S., Wera, S., Reinders, A., DeVirgilio, C., Wiemken, A., and Thevelein, J. M., Composition andfunctional analysis of the Saccharomyces cerevisiae trehalose synthasecomplex. J Biol Chem, 1998. 273(50): p. 33311-9].

However in E. coli, OtsA, which has TPS activity, and OtsB, which hasTPP activity, act independently. In Arabidopsis, more than tenhomologues have been found for both TPS and TPP genes. Functionalanalysis of the plant genes has concentrated on the Arabidopsis TPS1gene. Loss-of-function tps1 mutants are embryo lethal, and were notrescued by exogenous trehalose. The tps1 mutants can be rescued by aninducible TPS1 construct, but have reduced root growth, and continuedinduction is required for the transition to flowering. Plantsover-expressing AtTPS1 had increased dehydration (drought) tolerance andwere glucose- and ABA-insensitive [Avonce, N., Leyman, B.,Mascorro-Gallardo, J. O., Van Dijck, P., Thevelein, J. M., andIturriaga, G., The Arabidopsis trehalose-6-P synthase AtTPS1 gene is aregulator of glucose, abscisic acid, and stress signaling. PlantPhysiol, 2004. 136(3): p. 3649-59].

Arabidopsis also encodes a number of TPP homologs, encoding proteinswith a TPP domain, with highly conserved phosphatase motifs typical ofthis class of phosphohydrolases [Thaller, M. C., Schippa, S., andRossolini, G. M., Conserved sequence motifs among bacterial, eukaryotic,and archaeal phosphatases that define a new phosphohydrolasesuperfamily. Protein Sci, 1998. 7(7): p. 1647-52].

Arabidopsis TPP genes were first isolated by their ability to complementyeast tps2 mutants [Leyman, B., Van Dijck, P., and Thevelein, J. M., Anunexpected plethora of trehalose biosynthesis genes in Arabidopsisthaliana. Trends Plant Sci, 2001. 6(11): p. 510-3; Vogel, G.,Aeschbacher, R. A., Muller, J., Boller, T., and Wiemken, A.,Trehalose-6-phosphate phosphatases from Arabidopsis thaliana:identification by functional complementation of the yeast tps2 mutant.Plant J, 1998. 13(5): p. 673-83], though no other information isavailable on the biological functions of these genes.

Trehalose-6-phosphate has also recently emerged as a regulator of carbonmetabolism, and appears to act as an enhancer of photosynthetic capacitythrough interaction with sugar signaling pathways. Plantsover-expressing either the bacterial or yeast trehalose biosyntheticgenes have altered carbohydrate metabolism, and some morphologicaldefects. These phenotypes are thought to result from changes in carbonallocation between sink and source tissues and provoked speculation thattrehalose metabolism may be involved in sugar signaling. Paul et al.(Enhancing photosynthesis with sugar signals. Trends Plant Sci, 2001.6(5): p. 197-200) reasoned that T6P could be the signal that allowshexokinase to perceive carbon status, as is the case in yeast. Howeverthis is inconsistent with the fact that T6P is not an inhibitor ofArabidopsis hexokinases AtHXK1 and AtHXK2 in vitro, and reducinghexokinase activity did not rescue the growth of Arabidopsis tps1embryos. Nevertheless, an analysis of Arabidopsis plants over-expressingOtsA, OtsB and treC (trehalose phosphate hydrolase) also confirms aninvolvement of T6P in carbohydrate utilization and growth via control ofglycolysis.

Trehalose signaling also appears to play a similar role in monocots,since transgenic rice overexpressing E. coli OtsA and OtsB had increasedtrehalose accumulation, and this correlated with more solublecarbohydrates and a higher capacity for photosynthesis under both stressand non-stress conditions. These results are consistent with a role forthe trehalose pathway in modulating sugar sensing and carbohydratemetabolism.

In addition to T6P, other signaling steps in the plant trehalose pathwayhave been proposed. For example, the TPS protein interacts withregulatory 14-3-3 proteins, and this interaction may depend on thecellular sugar status. Trehalose itself may also act as a signal,although it is unlikely to act as an osmoprotectant as in microbes asthe concentration is very low. However, a possible target of trehaloseis ApL3, an ADP-glucose pyrophosphorylase, which is involved in starchbiosynthesis. These data suggest that trehalose interferes with carbonallocation to the sink tissues by inducing starch synthesis in sourcetissues.

While exact roles of trehalose and related sugars is not clearlyunderstood, sugars in general are thought of as signaling molecules oras global regulators of gene expression. In addition to their obviousmetabolic functions, sugars can act like hormones, or can modulatehormone signaling pathways.

SUMMARY OF THE INVENTION

The present invention includes:

An isolated polynucleotide comprising: (a) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:19,wherein expression of said polypeptide in a plant transformed with saidisolated polynucleotide results in alteration of the branching of thetassel, ear, or both, of said transformed plant when compared to acontrol plant not comprising said isolated polynucleotide; or (b) acomplement of the nucleotide sequence, wherein the complement and thenucleotide sequence consist of the same number of nucleotides and are100% complementary. Preferably, expression of said polypeptide resultsin a decrease in the branching of the tassel, ear, or both, and evenmore preferably, the plant is maize.

An isolated polynucleotide comprising: (a) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:19,wherein expression of said polypeptide in a plant transformed with saidisolated polynucleotide results in alteration of pollen shed of saidtransformed plant when compared to a control plant not comprising saidisolated polynucleotide; or (b) a complement of the nucleotide sequence,wherein the complement and the nucleotide sequence consist of the samenumber of nucleotides and are 100% complementary. Preferably, expressionof said polypeptide results in a decrease in pollen shed, and even morepreferably, the plant is maize.

An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide associated with branching of the tassel, ear, orboth, of a plant (preferably maize), wherein said polypeptide has anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19, or (b) a complement of the nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide associated with pollen shed of a plant(preferably maize), wherein said polypeptide has an amino acid sequenceof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:19, or (b) a complement of the nucleotide sequence, whereinthe complement and the nucleotide sequence consist of the same number ofnucleotides and are 100% complementary.

An isolated polynucleotide comprising: (a) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:19,wherein expression of said polypeptide in a plant exhibiting a ramosa3mutant phenotype results in an decrease of branching of the tassel, ear,or both of the plant; or (b) a complement of the nucleotide sequence,wherein the complement and the nucleotide sequence consist of the samenumber of nucleotides and are 100% complementary. Preferably, the plantis maize.

An isolated polynucleotide comprising: (a) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:19,wherein expression of said polypeptide in a plant exhibiting a ramosa3mutant phenotype results in an decrease of pollen shed of the plant; or(b) a complement of the nucleotide sequence, wherein the complement andthe nucleotide sequence consist of the same number of nucleotides andare 100% complementary. Preferably, the plant is maize.

An isolated polynucleotide comprising: (a) a nucleic acid sequenceencoding a polypeptide having trehalose-6-phosphate phosphataseactivity, wherein the polypeptide has an amino acid sequence of at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:19, 49, 68 or 69; or (b) a complement of the nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

Any recombinant DNA construct comprising a polynucleotide operablylinked to a promoter that is functional in said plant, wherein saidpolynucleotide comprises an isolated polynucleotide of the presentinvention.

A vector comprising a polynucleotide of the present invention.

A suppression DNA construct comprising a promoter functional in a plantoperably linked to (a) all or part of (i) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 50%sequence identity, or any integer up to and including 100% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:19, 49, 68 or 69, or (ii) a full complement of the nucleicacid sequence of (a)(i); (b) a region derived from all or part of asense strand or antisense strand of a target gene of interest, saidregion having a nucleic acid sequence of at least 50% sequence identity,based on the Clustal V method of alignment, when compared to said all orpart of a sense strand or antisense strand from which said region isderived, and wherein said target gene of interest encodes a RAMOSA3(RA3) polypeptide or a SISTER OF RAMOSA3 (SRA) polypeptide; or (c) anucleic acid sequence of at least 50% sequence identity, or any integerup to and including 100% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO:15, 18, 47, 48, or 67.The suppression DNA construct preferably comprises a cosuppressionconstruct, antisense construct, viral-suppression construct, hairpinsuppression construct, stem-loop suppression construct, double-strandedRNA-producing construct, RNAi construct, or small RNA construct (e.g.,an sRNA construct or an miRNA construct).

A plant comprising in its genome a recombinant DNA construct of thepresent invention.

A plant whose genome comprises a disruption (e.g., an insertion, such asa transposable element, or sequence mutation) of at least one gene(which may be heterologous or endogenous to the genome) encoding atleast one polypeptide selected from the group consisting of a RAMOSA3(RA3) polypeptide or a SISTER OF RAMOSA3 (SRA) polypeptide.

Any progeny of the above plants, and any seed obtained from the plant orits progeny. Progeny includes subsequent generations obtained byself-pollination or out-crossing of a plant. Progeny also includeshybrids and inbreds.

A method for altering branching of the tassel, ear, or both, of a plant,comprising: (a) introducing into a regenerable plant cell a recombinantDNA construct to produce a transformed plant cell, said recombinant DNAconstruct comprising a polynucleotide operably linked to a promoter thatis functional in a plant, wherein said polynucleotide encodes apolypeptide having an amino acid sequence of at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the ClustalV method of alignment, when compared to SEQ ID NO:19; and (b)regenerating a transgenic plant from said transformed plant cell,wherein said transgenic plant comprises in its genome said recombinantDNA construct and wherein said transgenic plant exhibits an alterationin branching of the tassel, ear, or both, when compared to a controlplant not comprising said recombinant DNA construct. The method mayfurther comprise (c) obtaining a progeny plant derived from saidtransgenic plant, wherein said progeny plant comprises in its genome therecombinant DNA construct. Preferably, the transgenic plant or progenythereof exhibits a decrease in branching of the tassel, ear, or both.

A method for altering pollen shed of a plant, comprising: (a)introducing into a regenerable plant cell a recombinant DNA construct toproduce a transformed plant cell, said recombinant DNA constructcomprising a polynucleotide operably linked to a promoter that isfunctional in a plant, wherein said polynucleotide encodes a polypeptidehaving an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO:19; and (b) regenerating atransgenic plant from said transformed plant cell, wherein saidtransgenic plant comprises in its genome said recombinant DNA constructand wherein said transgenic plant exhibits an alteration in pollen shed,when compared to a control plant not comprising said recombinant DNAconstruct. The method may further comprise (c) obtaining a progeny plantderived from said transgenic plant, wherein said progeny plant comprisesin its genome the recombinant DNA construct. Preferably, the transgenicplant or progeny thereof exhibits a decrease in pollen shed.

A method for altering trehalose-6-phosphate phosphatase activity in aplant, comprising: (a) introducing into a regenerable plant cell arecombinant DNA construct to produce a transformed plant cell, saidrecombinant DNA construct comprising a polynucleotide operably linked toa promoter that is functional in a plant, wherein said polynucleotideencodes a polypeptide having an amino acid sequence of at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:19, 49, 68 or 69; and (b) regenerating a transgenic plant fromsaid transformed plant cell, wherein said transgenic plant comprises inits genome said recombinant DNA construct and wherein said transgenicplant exhibits an alteration in trehalose-6-phosphate phosphataseactivity, when compared to a control plant not comprising saidrecombinant DNA construct. The method may further comprise (c) obtaininga progeny plant derived from said transgenic plant, wherein said progenyplant comprises in its genome the recombinant DNA construct. Preferably,the transgenic plant or progeny thereof exhibits an increase intrehalose-6-phosphate phosphatase activity.

A method for increasing environmental stress tolerance (preferablydrought tolerance) of a plant, comprising: (a) introducing into aregenerable plant cell a recombinant DNA construct to produce atransformed plant cell, said recombinant DNA construct comprising apolynucleotide operably linked to a promoter that is functional in aplant, wherein said polynucleotide encodes a polypeptide having an aminoacid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO:19, 49, 68 or 69; and (b)regenerating a transgenic plant from said transformed plant cell,wherein said transgenic plant comprises in its genome said recombinantDNA construct and wherein said transgenic plant exhibits an increase inenvironmental stress tolerance (preferably drought tolerance), whencompared to a control plant not comprising said recombinant DNAconstruct. The method may further comprise (c) obtaining a progeny plantderived from said transgenic plant, wherein said progeny plant comprisesin its genome the recombinant DNA construct.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1 shows the mature ear and tassel phenotypes of ra3 mutants.

FIG. 2 shows the SEM analysis of ra3 mutant ears. Wild type (B73) earsare shown in A-D, and ra3 ears in E-H. During the inflorescencetransition stage, there is no difference between B73 (A) and ra3 mutant(E) ears. In 2 mm long ears, some SPM converted to BM* (F, arrow 1). SMsin B73 produce a pair of glumes (C, arrows 2), but in ra3 they produceadditional glumes (G, arrows 2), and FMs were made inside the glumes(arrow 3). The SMs in wild type ears make 2 FMs inside the glumes (D,upper FM marked by arrow 3) and each floret has a lemma and palea (D,palea marked by arrow 4). The SMs in ra3 mutants make several FMs(arrows 3, H, palea marked by arrow 4) and later can convert to a BM*that makes glumes (H, arrows 2). Scale bars A, E 100 μm; B, F 500 μm; C,D 100 μm; G, H 200 μm.

FIG. 3 shows a schematic of wild type (wt) and ra3 ear development. BM*indicates a meristem which makes SPMs. SPM* indicates a meristem whichmakes SMs. SM* indicates meristem which makes FMs.

FIG. 4 shows a map of the RA3 locus. The position of the RA3 and relatedSRA loci on BAC clone c0387K01 is given in (A). Marker positions areshown as black dots and number of recombinants for each is shown. TheRA3 gene structure is given in (B). The shaded box immediately precedingthe start ATG codon and the shaded box immediately following the stopcodon each represent UTRs; the shaded boxes in between these UTRsindicate coding sequence; and “TATA” indicates the predicted TATA-box.

FIG. 5 shows the predicted RA3 protein structure and phylogeneticanalysis. The RA3 protein structure is shown in part A. The TPPsimilarity regions are labeled “2”, the phosphatase boxes are labeled“3”, and the non-conserved regions are labeled “1”. The neighbor joiningtree is shown in part B, using Phylogenetic Analysis Using Parsimony(“PAUP”; SWOFFORD D. L., (1993) J Gen Physiol 102:A9) of the conservedpart of the TPP region of RA3-like proteins in maize, rice andArabidopsis. Maize proteins are the following: RA3, SRA, ZmRA3L1-8. Riceproteins are the following: gi46390128, gi37806433, gi33146623,9631.m02649, 9683.m03678, 9634.m01158. Arabidopsis proteins are thefollowing: At5g51460, At1g35910, At2g22190, At4g39770, At5g10100,At5g65140.

FIGS. 6A, 6B, 6C and 6D show a sequence alignment of the following aminoacid sequences: (1) SEQ ID NO:19, for the corn RAMOSA3 polypeptide; (2)SEQ ID NO:49, for the corn SISTER OF RAMOSA3 polypeptide; (3) SEQ IDNO:51, for the rice trehalose-6-phosphate phosphatase polypeptidecorresponding to NCBI GI NO. 33146623; (4) SEQ ID NO:52, for theArabidopsis TPPA polypeptide corresponding to NCBI GI NO. 2944178; (5)SEQ ID NO:53, for the Arabidopsis TPPB polypeptide corresponding to NCBIGI NO. 2944180; (6) SEQ ID NO:54, for a corn trehalose-6-phosphatephosphatase polypeptide that is cited as SEQ ID NO:16 in U.S. PatentPublication 2004-0229364-A1; (7) SEQ ID NO:55, for a soybeantrehalose-6-phosphate phosphatase polypeptide that is cited as SEQ IDNO:20 in U.S. Patent Publication 2004-0229364-A1; (8) a truncatedversion of SEQ ID NO:52, in which the N-terminal 91 amino acids areremoved; this fragment was shown to have enzymatic activity (Vogel etal. (1998) Plant J 13(5):673-683); and (9) a truncated version of SEQ IDNO:53, in which the N-terminal 91 amino acids have been removed. Aconsensus sequence of 414 amino acids was generated and is numberedbelow these nine sequences. The amino acid positions for each sequenceis given to the left of each row, and to the right of the final row. Anasterisk above an amino acid residue indicates that the position istotally conserved among the given SEQ ID NOs, with respect to theArabidopsis thaliana AtTPPB sequence. Below the sequences are shown twodomains, A and B, that are conserved among trehalose-6-phosphatephosphatases, as described in Vogel et al. (1998) Plant J 13(5):673-683.The given sequence for each conserved domain is taken from theArabidopsis thaliana AtTPPB amino acid sequence at these positions.

FIG. 7 shows that RA3 expression is highest in developinginflorescences. In part (A) is shown the RA3 expression in 1 cm earprimordia from B73 (lane 1) and ra3 alleles. In ra3-ref (lane 2) andra3-feat (lane 3), very low expression was detected, while the level ofexpression in ra3-EV, ra3-NI and ra3-bre (lanes 4-6) was normal, exceptthat the ra3-NI transcript is slightly larger, as it has a smallinsertion (Table 2). The lower panel shows Ubiquitin (UBI) expression asa control. In part (B) is shown the RA3, SRA and Ubiquitin expressionduring wild type (B73) development. The mRNAs were extracted from root(R), vegetative apex (V), young leaves (L) and ear or tasselinflorescence primordia. The triangles represent increasinginflorescence size, from transition stage to ˜1.5 cm ears and tassels.RA3 expression peaks in 2-5 mm inflorescences.

FIG. 8 shows that RA3 expression is spatially restricted during eardevelopment. RA3 was first expressed at the base of SPMs (arrows, A).After that, RA3 expression enlarged to a cup-shaped domain at the baseof SPMs and SMs (arrows, B, C and D). Parts (B) and (C) are longitudinalmedian and glancing sections, respectively, and (D) is a transversesection. At later stages, RA3 was expressed at the boundary betweenupper and lower florets (arrows, E). No RA3 transcript was detected in ara3-ref ear (F), at similar stage to (B).

FIG. 9 shows phosphate release, measured as OD₆₀₀, following treatmentof various phosphorylated substrates. For each substrate, shown left toright, respectively, is the activity due to each of the followingproteins: 1) His-tagged RA3 full-length protein (SEQ ID NO:61); 2)His-tagged RA3 TPP-domain fragment (SEQ ID NO:62); 3) His-tagged RA3N-terminal fragment (SEQ ID NO:63); 4) His-tagged Mycobacteriumtuberculosis TPP (Edavana et al., Arch Biochem Biophys 426:250-257(2004)); 5) shrimp alkaline phosphatase (SAP; Roche Applied Science); 6)no protein.

FIG. 10 shows growth of a yeast tps2 mutant (yeast strain YSH6.106.-8C)transformed with an RA3 protein, RA3 TPP-domain fragment, a positivecontrol (Arabidopsis trehalose-6-phosphate phosphatase gene, AtTPPB),and a negative control (empty yeast vector). Transformed cells wereassayed for growth on selective media at 40.5° C. in the presence of 1MNaCl, as well as at 30° C.

SEQ ID NO:1 is the nucleotide sequence of the csu597 forward primer.

SEQ ID NO:2 is the nucleotide sequence of the csu597 reverse primer.

SEQ ID NO:3 is the nucleotide sequence of the umc1412 forward primer.

SEQ ID NO:4 is the nucleotide sequence of the umc1412 reverse primer.

SEQ ID NO:5 is the nucleotide sequence of the a14 forward primer.

SEQ ID NO:6 is the nucleotide sequence of the a14 reverse primer.

SEQ ID NO:7 is the nucleotide sequence of the n20 forward primer.

SEQ ID NO:8 is the nucleotide sequence of the n20 reverse primer.

SEQ ID NO:9 is the nucleotide sequence of the cb.g1E forward primer.

SEQ ID NO:10 is the nucleotide sequence of the cb.g1E reverse primer.

SEQ ID NO:11 is the nucleotide sequence of the NS346 primer.

SEQ ID NO:12 is the nucleotide sequence of the NS347 primer.

SEQ ID NO:13 is the nucleotide sequence of the NS362 primer.

SEQ ID NO:14 is the nucleotide sequence of the NS363 primer.

SEQ ID NO:15 is the genomic nucleotide sequence containing the RAMOSA3gene.

SEQ ID NO:16 is the nucleotide sequence of the NS432 forward primer usedto amplify the cDNA containing the RAMOSA3 open-reading frame.

SEQ ID NO:17 is the nucleotide sequence of the NS411 reverse primer usedto amplify the cDNA containing the RAMOSA3 open-reading frame.

SEQ ID NO:18 is the nucleotide sequence of the protein-coding regiondeduced from the PCR product obtained using the primers of SEQ ID NOs:16& 17 to amplify maize cDNA.

SEQ ID NO:19 is the amino acid sequence of the RAMOSA3 polypeptide.

SEQ ID NO:20 is the nucleotide sequence of the 5′ UTR-exon2 forwardprimer.

SEQ ID NO:21 is the nucleotide sequence of the 5′ UTR-exon2 reverseprimer.

SEQ ID NO:22 is the nucleotide sequence of the exon3-exon4 forwardprimer.

SEQ ID NO:23 is the nucleotide sequence of the exon3-exon4 reverseprimer.

SEQ ID NO:24 is the nucleotide sequence of the exon5-exon7 forwardprimer.

SEQ ID NO:25 is the nucleotide sequence of the exon5-exon7 reverseprimer.

SEQ ID NO:26 is the nucleotide sequence of the exon8-exon10 forwardprimer.

SEQ ID NO:27 is the nucleotide sequence of the exon8-exon10 reverseprimer.

SEQ ID NO:28 is the nucleotide sequence of the exon11-3′ UTR forwardprimer.

SEQ ID NO:29 is the nucleotide sequence of the exon11-3′ UTR reverseprimer.

SEQ ID NO:30 is the nucleotide sequence of a region in exon7 of thera3-ref mutant gene, that contains a 4 base pair insertion relative tothe sequence of SEQ ID NO:15.

SEQ ID NO:31 is the deduced amino acid sequence of the mutant ra3-refRAMOSA3 polypeptide, which has a frame-shift after amino acid 249,relative to SEQ ID NO:19, and a premature stop codon after 305 aminoacids.

SEQ ID NO:32 is the nucleotide sequence of an ILS-1-like transposonelement present in the 5′-UTR region of the ra3-fea1 mutant gene.

SEQ ID NO:33 is the nucleotide sequence of a region of exon7 in thera3-fea1 mutant gene, that contains a 4 base pair insertion relative tothe sequence of SEQ ID NO:15.

SEQ ID NO:34 is the deduced amino acid sequence of the mutant ra3-fea1RAMOSA3 polypeptide, which has a frame-shift after amino acid 258relative to SEQ ID NO:19, and a premature stop codon after 305 aminoacids.

SEQ ID NO:35 is the nucleotide sequence of a region of exon6 of thera3-EV mutant gene, that contains a 4 base pair insertion relative toSEQ ID NO:15.

SEQ ID NO:36 is the deduced amino acid sequence of the mutant ra3-EVRAMOSA3 polypeptide, which has a frame-shift after amino acid 224relative to SEQ ID NO:19, and a premature stop codon after 305 aminoacids.

SEQ ID NO:37 is the nucleotide sequence of a region of exon10 of thera3-NI mutant gene, that contains a 141 base pair insertion relative toSEQ ID NO:15.

SEQ ID NO:38 is the deduced amino acid sequence of the mutant ra3-NIRAMOSA3 polypeptide, which has a different amino acid sequence afteramino acid 333 relative to SEQ ID NO:19, and a premature stop codonafter 335 amino acids.

SEQ ID NO:39 is the nucleotide sequence of a region between exon6 andexon7 of the ra3-bre mutant gene, that contains a 10 base pair insertionrelative to SEQ ID NO:15.

SEQ ID NO:40 is the deduced amino acid sequence of the mutant ra3-breRAMOSA3 polypeptide, which has a frame-shift after amino acid 241 and apremature stop codon after 243 amino acids.

SEQ ID NO:41 is the nucleotide sequence of region of exon6 of the ra3-JLmutant gene, that contains a deletion and rearrangement relative to SEQID NO:15.

SEQ ID NO:42: is the deduced amino acid sequence of the mutant ra3-JLRAMOSA3 polypeptide, which has a different protein sequence after aminoacid 217 and a premature stop codon after 246 amino acids.

SEQ ID NO:43 is the nucleotide sequence of a region of exon6 of thera3-NS mutant gene, that contains a 2 base pair insertion relative toSEQ ID NO:15.

SEQ ID NO:44 is the deduced amino acid sequence of the mutant ra3-NSRAMOSA3 polypeptide, which has a frame-shift after amino acid 222 and apremature stop codon after 299 amino acids.

SEQ ID NO:45 is the amino acid sequence of the conserved “A-domain”phosphatase box, as described in Vogel et al. (Plant J. 13:673-683(1998)) and in U.S. Patent Publication 2004-0229364-A1, the entirecontents of which are herein incorporated by reference.

SEQ ID NO:46 is the amino acid sequence of the conserved “B-domain”phosphatase box, as described in Vogel et al. (Plant J. 13:673-683(1998)) and in U.S. Patent Publication 2004-0229364-A1.

SEQ ID NO:47 is the genomic nucleotide sequence containing the SISTER OFRAMOSA3 (SRA) gene.

SEQ ID NO:48 is the nucleotide sequence of the protein-coding region ofthe SRA gene.

SEQ ID NO:49 is the amino acid sequence of the SRA polypeptide.

SEQ ID NO:50 is a nucleotide sequence contained in the clonemy.cs1.pk0072.d4, which is a cDNA clone containing a fragment of the SRAgene.

SEQ ID NO:51 is the amino acid sequence of the ricetrehalose-6-phosphate phosphatase polypeptide corresponding to NCBI GINO. 33146623.

SEQ ID NO:52 is the amino acid sequence for the Arabidopsis AtTPPApolypeptide corresponding to NCBI GI NO. 2944178.

SEQ ID NO:53 is the amino acid sequence for the Arabidopsis AtTPPBpolypeptide corresponding to NCBI GI NO. 2944180.

SEQ ID NO:54 is the amino acid sequence for a corn trehalose-6-phosphatephosphatase polypeptide that is cited as SEQ ID NO:16 in U.S. PatentPublication 2004-0229364-A1, the entire contents of which are hereinincorporated by reference.

SEQ ID NO:55 is the amino acid sequence for a soybeantrehalose-6-phosphate phosphatase polypeptide that is cited as SEQ IDNO:20 in U.S. Patent Publication 2004-0229364-A1.

SEQ ID NO:56 is the nucleotide sequence of the NS487 primer.

SEQ ID NO:57 is the nucleotide sequence of the NS429 primer.

SEQ ID NO:58 is the nucleotide sequence of the NS483 primer.

SEQ ID NO:59 is the nucleotide sequence of the NS485 primer.

SEQ ID NO:60 is the nucleotide sequence of the NS488 primer.

SEQ ID NO:61 is the amino acid sequence of the His-tagged RA3 proteinproduced in E. coli. SEQ ID NO:61 consists of a 37-aa N-terminal regionthat contains six consecutive histidine residues, followed by the 361-aaresidues of the RA3 protein (SEQ ID NO:19).

SEQ ID NO:62 is the amino acid sequence of the His-tagged RA3 TPP-domainfragment produced in E. coli. SEQ ID NO:62 consists of a 35-aaN-terminal region that contains six consecutive histidine residues,followed by amino acid residues 78-361 of the RA3 protein (SEQ IDNO:19).

SEQ ID NO:63 is the amino acid sequence of the His-tagged RA3 N-terminalfragment produced in E. coli. SEQ ID NO:63 consists of a 34-aaN-terminal region that contains six consecutive histidine residues,followed by amino acid residues 1-78 of the RA3 protein (SEQ ID NO:19).

SEQ ID NO:64 is the nucleotide sequence of the NS489 primer.

SEQ ID NO:65 is the nucleotide sequence of the NS490 primer.

SEQ ID NO:66 is the nucleotide sequence of the NS500 primer.

SEQ ID NO:67 is the nucleotide sequence of the DNA fragment encoding aRA3 TPP-domain fragment, that was shown to rescue growth of the yeasttps2 mutant at the non-permissive temperature. SEQ ID NO:67 consists ofan ATG start codon followed by nucleotides 235-1086 of SEQ ID NO:18.

SEQ ID NO:68 is the amino acid sequence of the RA3 TPP-domain fragmentencoded by SEQ ID NO:67. SEQ ID NO:68 consists of a start methionineresidue followed by amino acid residues 79-361 of SEQ ID NO:19.

SEQ ID NO:69 is the amino acid sequence of the SRA TPP-domain fragment,and corresponds to amino acids 76-370 of SEQ ID NO:49.

SEQ ID NO:70 corresponds to amino acids 92-385 of SEQ ID NO:52(Arabidopsis AtTPPA); this polypeptide fragment has been shown to haveenzymatic activity (Vogel et al. (1998) Plant J 13(5):673-683).

SEQ ID NO:71 corresponds to amino acids 92-374 of SEQ ID NO:53(Arabidopsis AtTPPB).

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J.219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822. The sequencedescriptions and Sequence Listing attached hereto comply with the rulesgoverning nucleotide and/or amino acid sequence disclosures in patentapplications as set forth in 37 C.F.R. §1.821-1.825.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The term “RAMOSA3 (RA3) gene” is a gene of the present invention andrefers to a non-heterologous form of a full-length RAMOSA3 (RA3)polynucleotide. In a preferred embodiment, the RAMOSA3 gene comprisesSEQ ID NO:15 or 18.

“RAMOSA3 (RA3) polypeptide” refers to a polypeptide of the presentinvention and may comprise one or more amino acid sequences, inglycosylated or non-glycosylated form. In a preferred embodiment, theRAMOSA3 (RA3) polypeptide comprises SEQ ID NO:19. A “RAMOSA3 (RA3)protein” comprises a RAMOSA3 (RA3) polypeptide.

“SISTER OF RAMOSA3 (SRA) gene” is a gene of the present invention andrefers to a non-heterologous genomic form of a full-length SISTER OFRAMOSA3 (SRA) polynucleotide. In a preferred embodiment, the SISTER OFRAMOSA3 (SRA) gene comprises SEQ ID NO:47 or 48.

“SISTER OF RAMOSA3 (SRA) polypeptide” refers to a polypeptide of thepresent invention and may comprise one or more amino acid sequences, inglycosylated or non-glycosylated form. In a preferred embodiment, theSISTER OF RAMOSA3 (SRA) polypeptide comprises SEQ ID NO:49. A “SISTER OFRAMOSA3 (SRA) protein” comprises a SISTER OF RAMOSA3 (SRA) polypeptide.

“Transgenic” includes any cell, cell line, callus, tissue, plant part orplant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. Preferably, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably and is a polymer of RNAor DNA that is single or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from amRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product have been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

“Allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome. Different alleles of a gene differ in their DNAsequence. When the alleles present at a given locus on a pair ofhomologous chromosomes in a diploid plant are the same that plant ishomozygous at that locus. If the alleles present at a given locus on apair of homologous chromosomes in a diploid plant differ that plant isheterozygous at that locus. If a transgene is present on one of a pairof homologous chromosomes in a diploid plant that plant is hemizygous atthat locus.

“Contig” refers to a nucleotide sequence that is assembled from two ormore constituent nucleotide sequences that share common or overlappingregions of sequence homology. For example, the nucleotide sequences oftwo or more nucleic acid fragments can be compared and aligned in orderto identify common or overlapping sequences. Where common or overlappingsequences exist between two or more nucleic acid fragments, thesequences (and thus their corresponding nucleic acid fragments) can beassembled into a single contiguous nucleotide sequence.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well-established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

The term “amplified” means the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequences as atemplate. Amplification systems include the polymerase chain reaction(PCR) system, ligase chain reaction (LCR) system, nucleic acid sequencebased amplification (NASBA, Cangene, Mississauga, Ontario), Q-BetaReplicase systems, transcription-based amplification system (TAS), andstrand displacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “chromosomal location” includes reference to a length of achromosome which may be measured by reference to the linear segment ofDNA which it comprises. The chromosomal location can be defined byreference to two unique DNA sequences, i.e., markers.

The term “marker” includes reference to a locus on a chromosome thatserves to identify a unique position on the chromosome. A “polymorphicmarker” includes reference to a marker which appears in multiple forms(alleles) such that different forms of the marker, when they are presentin a homologous pair, allow transmission of each of the chromosomes inthat pair to be followed. A genotype may be defined by use of one or aplurality of markers.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Unless stated otherwise, multiple alignment of the sequences providedherein were performed using the Clustal V method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments and calculation of percent identity of protein sequencesusing the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAPPENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Unless otherwise stated, “BLAST” sequence identity/similarity valuesprovided herein refer to the value obtained using the BLAST 2.0 suite ofprograms using default parameters (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPS) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPS containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=A, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad.Sci. USA 89:10915 (1989)).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Turning now to preferred embodiments:

The present invention includes isolated polynucleotides.

In one preferred embodiment, an isolated polynucleotide comprises: (a) anucleic acid sequence encoding a polypeptide having an amino acidsequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:19, wherein expression of said polypeptide in aplant transformed with said isolated polynucleotide results inalteration of the branching of the tassel, ear, or both, of saidtransformed plant when compared to a control plant not comprising saidisolated polynucleotide; or (b) a complement of the nucleotide sequence,wherein the complement and the nucleotide sequence consist of the samenumber of nucleotides and are 100% complementary. Preferably, expressionof said polypeptide results in a decrease in the branching of thetassel, ear, or both, and even more preferably, the plant is maize.

In another preferred embodiment, an isolated polynucleotide comprises:(a) a nucleic acid sequence encoding a polypeptide having an amino acidsequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:19, wherein expression of said polypeptide in aplant transformed with said isolated polynucleotide results inalteration of pollen shed of said transformed plant when compared to acontrol plant not comprising said isolated polynucleotide; or (b) acomplement of the nucleotide sequence, wherein the complement and thenucleotide sequence consist of the same number of nucleotides and are100% complementary. Preferably, expression of said polypeptide resultsin a decrease in pollen shed, and even more preferably, the plant ismaize.

In another preferred embodiment, an isolated polynucleotide comprises:(a) a nucleotide sequence encoding a polypeptide associated withbranching of the tassel, ear, or both, of a plant (preferably maize),wherein said polypeptide has an amino acid sequence of at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:19, or (b)a complement of the nucleotide sequence, wherein the complement and thenucleotide sequence consist of the same number of nucleotides and are100% complementary.

In another preferred embodiment, an isolated polynucleotide comprises:(a) a nucleotide sequence encoding a polypeptide associated with pollenshed of a plant (preferably maize), wherein said polypeptide has anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19, or (b) a complement of the nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

A polypeptide is “associated with branching” or “associated with pollenshed” in that the absence of the polypeptide in a plant results in anincrease in branching or pollen shed of the plant when compared to aplant that expresses the polypeptide.

In another preferred embodiment, an isolated polynucleotide comprises:(a) a nucleic acid sequence encoding a polypeptide having an amino acidsequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:19, wherein expression of said polypeptide in aplant exhibiting a ramosa3 mutant phenotype results in an decrease ofbranching of the tassel, ear, or both of the plant; or (b) a complementof the nucleotide sequence, wherein the complement and the nucleotidesequence consist of the same number of nucleotides and are 100%complementary. Preferably, the plant is maize.

In another preferred embodiment, an isolated polynucleotide comprises:(a) a nucleic acid sequence encoding a polypeptide having an amino acidsequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:19, wherein expression of said polypeptide in aplant exhibiting a ramosa3 mutant phenotype results in an decrease ofpollen shed of the plant; or (b) a complement of the nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary. Preferably,the plant is maize.

In another preferred embodiment, an isolated polynucleotide comprises:(a) a nucleic acid sequence encoding a polypeptide havingtrehalose-6-phosphate phosphatase activity, wherein the polypeptide hasan amino acid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO:19, 49, 68 or 69; or (b)a complement of the nucleotide sequence, wherein the complement and thenucleotide sequence consist of the same number of nucleotides and are100% complementary. Preferably, when compared to SEQ ID NO:68 or 69, thepolypeptide has an amino acid sequence of at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on theClustal V method of alignment.

The present invention also includes isolated polypeptides.

In a preferred embodiment, an isolated polypeptide comprises an aminoacid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:19, wherein expression of said polypeptide in aplant transformed with an isolated polynucleotide encoding saidpolypeptide results in alteration of the branching of the tassel, ear,or both, of the plant, when compared to a control plant not comprisingsaid expressed polypeptide. Preferably, expression of said polypeptideresults in a decrease in the branching, and even more preferably, theplant is maize.

In another preferred embodiment, an isolated polypeptide comprises anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19, wherein expression of said polypeptide ina plant transformed with an isolated polynucleotide encoding saidpolypeptide results in alteration of pollen shed of the plant, whencompared to a control plant not comprising said expressed polypeptide.Preferably, expression of said polypeptide results in a decrease in thepollen shed, and even more preferably, the plant is maize.

In another preferred embodiment, an isolated polypeptide comprises anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19, wherein expression of said polypeptide ina plant exhibiting a ramosa3 mutant phenotype results in an decrease ofbranching of the tassel, ear, or both, of the plant. Preferably, theplant is maize.

In another preferred embodiment, an isolated polypeptide comprises anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19, wherein expression of said polypeptide ina plant exhibiting a ramosa3 mutant phenotype results in an decrease ofpollen shed of the plant. Preferably, the plant is maize.

Another preferred embodiment included within the present invention is anisolated polypeptide associated with branching of the tassel, ear, orboth, of a plant (preferably maize), wherein said polypeptide has anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19. Another preferred embodiment is anisolated polypeptide associated with pollen shed of a plant (preferablymaize), wherein said polypeptide has an amino acid sequence of at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, basedon the Clustal V method of alignment, when compared to SEQ ID NO:19.

Still another preferred embodiment is an isolated polypeptide havingtrehalose-6-phosphate phosphatase activity, wherein the polypeptide hasan amino acid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO:19, 49, 68 or 69.

It is understood, as those skilled in the art will appreciate, that theinvention encompasses more than the specific exemplary sequences.Alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not effectthe functional properties of the encoded polypeptide, are well known inthe art. For example, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue, such as glycine, or a more hydrophobic residue,such as valine, leucine, or isoleucine. Similarly, changes which resultin substitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, can also be expected to produce afunctionally equivalent product. Nucleotide changes which result inalteration of the N-terminal and C-terminal portions of the polypeptidemolecule would also not be expected to alter the activity of thepolypeptide. Each of the proposed modifications is well within theroutine skill in the art, as is determination of retention of biologicalactivity of the encoded products.

The present invention also includes a recombinant DNA constructcomprising a polynucleotide operably linked to a promoter that isfunctional in said plant, wherein said polynucleotide comprises anisolated polynucleotide of the present invention, such as a preferredpolynucleotide as described above.

In one preferred embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to a promoter that is functional in saidplant, wherein said polynucleotide encodes a polypeptide having an aminoacid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% sequence identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO:19, 49, 68 or 69. Preferably, whencompared to SEQ ID NO:68 or 69, the polypeptide has an amino acidsequence of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity, based on the Clustal V method of alignment.

In another preferred embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to a promoter that is functional in saidplant, wherein said polynucleotide encodes a polypeptide having an aminoacid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:19.

The present invention also includes a suppression DNA construct.

A suppression construct preferably comprises a promoter functional in aplant operably linked to (a) all or part of (i) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 50%sequence identity, or any integer up to and including 100% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO:19, 49, 68 or 69, or (ii) a full complement of the nucleicacid sequence of (a)(i); (b) a region derived from all or part of asense strand or antisense strand of a target gene of interest, saidregion having a nucleic acid sequence of at least 50% sequence identity,based on the Clustal V method of alignment, when compared to said all orpart of a sense strand or antisense strand from which said region isderived, and wherein said target gene of interest encodes a RAMOSA3(RA3) polypeptide or a SISTER OF RAMOSA3 (SRA) polypeptide; or (c) anucleic acid sequence of at least 50% sequence identity, or any integerup to and including 100% identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO:15, 18, 47, 48 or 67. Thesuppression DNA construct preferably comprises a cosuppressionconstruct, antisense construct, viral-suppression construct, hairpinsuppression construct, stem-loop suppression construct, double-strandedRNA-producing construct, RNAi construct, or small RNA construct (e.g.,an sRNA construct or an miRNA construct).

As used herein, “suppression DNA construct” is a recombinant DNAconstruct which when transformed or stably integrated into the genome ofthe plant, results in “silencing” of a target gene in the plant. Thetarget gene may be endogenous or transgenic to the plant. “Silencing,”as used herein with respect to the target gene, refers generally to thesuppression of levels of mRNA or protein/enzyme expressed by the targetgene, and/or the level of the enzyme activity or protein functionality.The term “suppression” includes lower, reduce, decline, decrease,inhibit, eliminate and prevent. “Silencing” or “gene silencing” does notspecify mechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches, and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50% or anyinteger between 50% and 100% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as sRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocks theexpression of a target isolated nucleic acid fragment (U.S. Pat. No.5,107,065). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target protein. “Sense” RNArefers to RNA transcript that includes the mRNA and can be translatedinto protein within a cell or in vitro. Cosuppression constructs inplants have been previously designed by focusing on overexpression of anucleic acid sequence having homology to a native mRNA, in the senseorientation, which results in the reduction of all RNA having homologyto the overexpressed sequence (see Vaucheret et al. (1998) Plant J.16:651-659; and Gura (2000) Nature 404:804-808).

A number of promoters can be used in recombinant DNA constructs andsuppression DNA constructs of the present invention. The promoters canbe selected based on the desired outcome, and may include constitutive,tissue-specific, inducible, or other promoters for expression in thehost organism.

High level, constitutive expression of the candidate gene under controlof the 35S promoter may have pleiotropic affects. However, tissuespecific and/or stress-specific expression may eliminate undesirableaffects but retain the ability to enhance drought tolerance. This affecthas been observed in Arabidopsis (Kasuga et al. (1999) NatureBiotechnol. 17:287-91). As such, candidate gene efficacy may be testedwhen driven by different promoters.

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812(1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In choosing a promoter to use in the methods of the invention, it may bedesirable to use a tissue-specific or developmentally regulatedpromoter. A preferred tissue-specific or developmentally regulatedpromoter is a DNA sequence which regulates the expression of a DNAsequence selectively in the cells/tissues of a plant critical to tasseldevelopment, seed set, or both, and limits the expression of such a DNAsequence to the period of tassel development or seed maturation in theplant. Any identifiable promoter may be used in the methods of thepresent invention which causes the desired temporal and spatialexpression.

Promoters which are seed or embryo specific and may be useful in theinvention include soybean Kunitz trysin inhibitor (Kti3, Jofuku andGoldberg, Plant Cell 1:1079-1093 (1989)), patatin (potato tubers)(Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29), convicilin, vicilin,and legumin (pea cotyledons) (Rerie, W. G., et al. (1991) Mol. Gen.Genet. 259:149-157; Newbigin, E. J., et al. (1990) Planta 180:461-470;Higgins, T. J. V., et al. (1988) Plant. Mol. Biol. 11:683-695), zein(maize endosperm) (Schemthaner, J. P., et al. (1988) EMBO J.7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al.(1985) Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324), phytohemagglutinin(bean cotyledon) (Voelker, T. et al. (1987) EMBO J. 6:3571-3577),B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al. (1988)EMBO J. 7:297-302), glutelin (rice endosperm), hordein (barleyendosperm) (Marris, C., et al. (1988) Plant Mol. Biol. 10:359-366),glutenin and gliadin (wheat endosperm) (Colot, V., et al. (1987) EMBO J.6:3559-3564), and sporamin (sweet potato tuberous root) (Hattori, T., etal. (1990) Plant Mol. Biol. 14:595-604). Promoters of seed-specificgenes operably linked to heterologous coding regions in chimeric geneconstructions maintain their temporal and spatial expression pattern intransgenic plants. Such examples include Arabidopsis thaliana 2S seedstorage protein gene promoter to express enkephalin peptides inArabidopsis and Brassica napus seeds (Vanderkerckhove et al.,Bio/Technology 7:L929-932 (1989)), bean lectin and bean beta-phaseolinpromoters to express luciferase (Riggs et al., Plant Sci. 63:47-57(1989)), and wheat glutenin promoters to express chloramphenicol acetyltransferase (Colot et al., EMBO J. 6:3559-3564 (1987)).

Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical, and/or developmental signals.Inducible or regulated promoters include, for example, promotersregulated by light, heat, stress, flooding or drought, phytohormones,wounding, or chemicals such as ethanol, jasmonate, salicylic acid, orsafeners.

Promoters which are timed to stress include the following: 1) the RD29Apromoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91); 2) barleypromoter, B22E; expression of B22E is specific to the pedicel indeveloping maize kernels (“Primary Structure of a Novel Barley GeneDifferentially Expressed in Immature Aleurone Layers”. Klemsdae, S. S.et al., Mol. Gen. Genet. 228(1/2):9-16 (1991)); and 3) maize promoter,Zag2 (“Identification and molecular characterization of ZAG1, the maizehomolog of the Arabidopsis floral homeotic gene AGAMOUS”, Schmidt, R. J.et al., Plant Cell 5(7):729-737 (1993)). Zag2 transcripts can bedetected 5 days prior to pollination to 7 to 8 DAP, and directsexpression in the carpel of developing female inflorescences and CimIwhich is specific to the nucleus of developing maize kernels. CimItranscript is detected 4 to 5 days before pollination to 6 to 8 DAP.Other useful promoters include any promoter which can be derived from agene whose expression is maternally associated with developing femaleflorets.

Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of some variation may have identical promoter activity.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Newpromoters of various types useful in plant cells are constantly beingdiscovered; numerous examples may be found in the compilation byOkamuro, J. K., and Goldberg, R. B., Biochemistry of Plants 15:1-82(1989).

Particularly preferred promoters may include: RIP2, mLIP15, ZmCOR1,Rab17, CaMV 35S, RD29A, SAM synthetase, ubiquitin, CaMV 19S, nos, Adh,sucrose synthase, R-allele, or root cell promoter.

Recombinant DNA constructs and suppression DNA constructs of the presentinvention may also include other regulatory sequences, including but notlimited to, translation leader sequences, introns, and polyadenylationrecognition sequences. In another preferred embodiment of the presentinvention, a recombinant DNA construct of the present invention furthercomprises an enhancer or silencer.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol.Cell. Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200(1987). Such intron enhancement of gene expression is typically greatestwhen placed near the 5′ end of the transcription unit. Use of maizeintrons Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in theart. See generally, The Maize Handbook, Chapter 116, Freeling andWalbot, Eds., Springer, New York (1994).

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

A translation leader sequence is a DNA sequence located between thepromoter sequence of a gene and the coding sequence. The translationleader sequence is present in the fully processed mRNA upstream of thetranslation start sequence. The translation leader sequence may affectprocessing of the primary transcript to mRNA, mRNA stability ortranslation efficiency. Examples of translation leader sequences havebeen described (Turner, R. and Foster, G. D. (1995) MolecularBiotechnology 3:225).

Any plant can be selected for the identification of regulatory sequencesand genes to be used in creating recombinant DNA constructs andsuppression DNA constructs of the present invention. Examples ofsuitable plant targets for the isolation of genes and regulatorysequences would include but are not limited to alfalfa, apple, apricot,Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley,beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage,canola, cantaloupe, carrot, cassaya, castorbean, cauliflower, celery,cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee,corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive,escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit,honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblollypine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm,oil seed rape, okra, olive, onion, orange, an ornamental plant, palm,papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon,pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin,quince, radiata pine, radiscchio, radish, rapeseed, raspberry, rice,rye, sorghum, Southern pine, soybean, spinach, squash, strawberry,sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea,tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat,yams, and zucchini. Particularly preferred plants for the identificationof regulatory sequences are Arabidopsis, corn, wheat, soybean, andcotton.

The present invention also includes a plant comprising in its genome arecombinant DNA construct of the present invention (such as a preferredconstruct discussed above). Preferably, the recombinant DNA construct isstably integrated into the genome of the plant.

The present invention also includes a plant whose genome comprises adisruption (e.g., an insertion, such as a transposable element, orsequence mutation) of at least one gene (which may be heterologous orendogenous to the genome) encoding a polypeptide selected from the groupconsisting of a RAMOSA3 (RA3) polypeptide or a SISTER OF RAMOSA3 (SRA)polypeptide.

Also included in the present invention are any progeny of a plant of thepresent invention, and any seed obtained from such a plant or itsprogeny. Progeny includes subsequent generations obtained byself-pollination or out-crossing of a plant. Progeny also includeshybrids and inbreds. Preferably, in hybrid seed propagated crops, maturetransgenic plants can be self-crossed to produce a homozygous inbredplant. The inbred plant produces seed containing the newly introducedrecombinant DNA construct. These seeds can be grown to produce plantsthat would exhibit increased drought tolerance, or used in a breedingprogram to produce hybrid seed, which can be grown to produce plantsthat would exhibit increased drought tolerance. Preferably, the seedsare maize.

Preferably, a plant of the present invention is a monocotyledonous ordicotyledonous plant, more preferably, a maize or soybean plant, evenmore preferably a maize plant, such as a maize hybrid plant or a maizeinbred plant. The plant may also be sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley or millet.

Particularly preferred embodiments of plants of the present inventioninclude:

1. A plant (preferably maize or soybean, more preferably a plantexhibiting a ramosa3 mutant phenotype) comprising in its genome arecombinant DNA construct comprising an isolated polypeptide having anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19, wherein said plant exhibits alteration(preferably a decrease) of branching of the tassel, ear, or both, ofsaid plant when compared to a control plant not comprising saidrecombinant DNA construct.

2. A plant (preferably maize or soybean, more preferably a plantexhibiting a ramosa3 mutant phenotype) comprising in its genome arecombinant DNA construct comprising an isolated polypeptide having anamino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% sequence identity, based on the Clustal V method of alignment,when compared to SEQ ID NO:19, wherein said plant exhibits alteration(preferably a decrease) of pollen shed of said plant when compared to acontrol plant not comprising said recombinant DNA construct.

3. A plant (preferably maize or soybean, more preferably a plantexhibiting a ramosa3 mutant phenotype) comprising in its genome arecombinant DNA construct comprising an isolated polypeptide having anamino acid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% sequence identity, based on the Clustal V methodof alignment, when compared to SEQ ID NO:19, 49, 68 or 69, wherein saidplant exhibits increased trehalose-6-phosphate phosphatase activity whencompared to a control plant not comprising said recombinant DNAconstruct.

4. A plant (preferably maize or soybean, more preferably a plantexhibiting a ramosa3 mutant phenotype) comprising in its genome arecombinant DNA construct comprising an isolated polypeptide having anamino acid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% sequence identity, based on the Clustal V methodof alignment, when compared to SEQ ID NO:19, 49, 68 or 69, wherein saidplant exhibits increased environmental stress tolerance (preferablydrought tolerance) when compared to a control plant not comprising saidrecombinant DNA construct.

5. A plant (preferably maize or soybean) comprising in its genome:

a suppression DNA construct comprising a promoter functional in a plantoperably linked to:

-   -   (a) all or part of (i) a nucleic acid sequence encoding a        polypeptide having an amino acid sequence of at least 50%        sequence identity, or any integer up to and including 100%        sequence identity, based on the Clustal V method of alignment,        when compared to SEQ ID NO:19, 49, 68 or 69, or (ii) a full        complement of the nucleic acid sequence of (a)(i); or    -   (b) a region derived from all or part of a sense strand or        antisense strand of a target gene of interest, said region        having a nucleic acid sequence of at least 50% sequence        identity, based on the Clustal V method of alignment, when        compared to said all or part of a sense strand or antisense        strand from which said region is derived, and wherein said        target gene of interest encodes a polypeptide selected from the        group consisting of a RAMOSA3 (RA3) polypeptide or a SISTER OF        RAMOSA3 (SRA) polypeptide,

and wherein said plant exhibits increased branching of the tassel, ear,or both, and/or increased pollen shed, and/or reducedtrehalose-6-phosphate phosphatase activity when compared to a controlplant not comprising said suppression DNA construct. The suppression DNAconstruct preferably comprises a cosuppression construct, antisenseconstruct, viral-suppression construct, hairpin suppression construct,stem-loop suppression construct, double-stranded RNA-producingconstruct, RNAi construct, or small RNA construct (e.g., an sRNAconstruct or an miRNA construct).

6. A plant (preferably maize or soybean) whose genome comprises adisruption (e.g., an insertion, such as a transposable element, orsequence mutation) of at least one gene (which may be heterologous orendogenous to the genome) encoding a polypeptide selected from the groupconsisting of a RAMOSA3 (RA3) polypeptide or a SISTER OF RAMOSA3 (SRA)polypeptide, wherein said disruption results in said plant exhibitingincreased branching of the tassel, ear, or both, and/or increased pollenshed, and/or reduced trehalose-6-phosphate phosphatase activity whencompared to a control plant not comprising said disruption. Preferably,relative to SEQ ID NO:15, the disruption comprises any one of theinsertions shown in SEQ ID NOs:30, 32, 33, 35, 37, 39 or 43, or thedeletion and rearrangement shown in SEQ ID NO:41.

7. Any progeny of the above plants 1-6, any seeds of the above plants1-6, any seeds of progeny of the above plants 1-6, and cells from any ofthe above plants 1-6 and progeny.

The present invention also includes methods for altering branching ofthe tassel, ear, or both, of a plant; methods for altering pollen shedof a plant; methods for altering trehalose-6-phosphate phosphataseactivity in a plant; and methods for increasing environmental stresstolerance (preferably drought tolerance) in a plant. Preferably, theplant is a monocotyledonous or dicotyledonous plant, more preferably, amaize or soybean plant, even more preferably a maize plant. The plantmay also be sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley or millet.

In one preferred embodiment, a method for altering branching of thetassel, ear, or both, of a plant, comprises: (a) introducing into aregenerable plant cell a recombinant DNA construct to producetransformed plant cells, said recombinant DNA construct comprising apolynucleotide operably linked to a promoter that is functional in aplant, wherein said polynucleotide encodes a polypeptide having an aminoacid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:19; and (b) regenerating a transgenic plant fromsaid transformed plant cell, wherein said transgenic plant comprises inits genome said recombinant DNA construct and wherein said transgenicplant exhibits an alteration in branching of the tassel, ear, or both,when compared to a control plant not comprising said recombinant DNAconstruct. The method may further comprise (c) obtaining a progeny plantderived from said transgenic plant, wherein said progeny plant comprisesin its genome the recombinant DNA construct. Preferably, the transgenicplant or progeny thereof exhibits a decrease in branching of the tassel,ear, or both.

In another preferred embodiment, a method for altering pollen shed of aplant, comprises: (a) introducing into a regenerable plant cell arecombinant DNA construct to produce transformed plant cells, saidrecombinant DNA construct comprising a polynucleotide operably linked toa promoter that is functional in a plant, wherein said polynucleotideencodes a polypeptide having an amino acid sequence of at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO:19; and(b) regenerating a transgenic plant from said transformed plant cell,wherein said transgenic plant comprises in its genome said recombinantDNA construct and wherein said transgenic plant exhibits an alterationin pollen shed, when compared to a control plant not comprising saidrecombinant DNA construct. The method may further comprise (c) obtaininga progeny plant derived from said transgenic plant, wherein said progenyplant comprises in its genome the recombinant DNA construct. Preferably,the transgenic plant or progeny thereof exhibits a decrease in pollenshed.

Another preferred method of the present invention is a method foraltering trehalose-6-phosphate phosphatase activity in a plant,comprising: (a) introducing into a regenerable plant cell a recombinantDNA construct to produce transformed plant cells, said recombinant DNAconstruct comprising a polynucleotide operably linked to a promoter thatis functional in a plant, wherein said polynucleotide encodes apolypeptide having an amino acid sequence of at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, basedon the Clustal V method of alignment, when compared to SEQ ID NO:19, 49,68 or 69; and (b) regenerating a transgenic plant from said transformedplant cell, wherein said transgenic plant comprises in its genome saidrecombinant DNA construct and wherein said transgenic plant exhibits analteration in trehalose-6-phosphate phosphatase activity, when comparedto a control plant not comprising said recombinant DNA construct. Themethod may further comprise (c) obtaining a progeny plant derived fromsaid transgenic plant, wherein said progeny plant comprises in itsgenome the recombinant DNA construct. Preferably, the transgenic plantor progeny thereof exhibits an increase in trehalose-6-phosphatephosphatase activity.

In another preferred embodiment, a method for increasing environmentalstress tolerance (preferably drought tolerance) of a plant, comprises:(a) introducing into a regenerable plant cell a recombinant DNAconstruct to produce transformed plant cells, said recombinant DNAconstruct comprising a polynucleotide operably linked to a promoter thatis functional in a plant, wherein said polynucleotide encodes apolypeptide having an amino acid sequence of at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, basedon the Clustal V method of alignment, when compared to SEQ ID NO:19, 49,68 or 69; and (b) regenerating a transgenic plant from said transformedplant cell, wherein said transgenic plant comprises in its genome saidrecombinant DNA construct and wherein said transgenic plant exhibits anincrease in environmental stress tolerance (preferably droughttolerance), when compared to a control plant not comprising saidrecombinant DNA construct. The method may further comprise (c) obtaininga progeny plant derived from said transgenic plant, wherein said progenyplant comprises in its genome the recombinant DNA construct.

In yet another preferred embodiment, a method for increasing branchingof the tassel, ear, or both, and/or increasing pollen shed, and/orreducing trehalose-6-phosphate phosphatase activity a plant, comprises:(a) introducing into a regenerable plant cell a suppression DNAconstruct to produce transformed plant cells, said suppression DNAconstruct comprising a promoter functional in a plant operably linked to(i) all or part of (A) a nucleic acid sequence encoding a polypeptidehaving an amino acid sequence of at least 50% sequence identity, or anyinteger up to and including 100% sequence identity, based on the ClustalV method of alignment, when compared to SEQ ID NO:19, 49, 68 or 69, or(B) a full complement of the nucleic acid sequence of (i)(A), or (ii) aregion derived from all or part of a sense strand or antisense strand ofa target gene of interest, said region having a nucleic acid sequence ofat least 50% sequence identity, based on the Clustal V method ofalignment, when compared to said all or part of a sense strand orantisense strand from which said region is derived, and wherein saidtarget gene of interest encodes a polypeptide selected from the groupconsisting of a RAMOSA3 (RA3) polypeptide or a SISTER OF RAMOSA3 (SRA)polypeptide; and (b) regenerating a transgenic plant from saidtransformed plant cell, wherein said transgenic plant comprises in itsgenome said suppression DNA construct and wherein said transgenic plantexhibits increased branching of the tassel, ear, or both, and/orincreased pollen shed, and/or reduced trehalose-6-phosphate phosphataseactivity when compared to a control plant not comprising saidsuppression DNA construct. The method may further comprise (c) obtaininga progeny plant derived from said transgenic plant, wherein said progenyplant comprises in its genome the suppression DNA construct.

The introduction of recombinant DNA constructs of the present inventioninto plants may be carried out by any suitable technique, including butnot limited to direct DNA uptake, chemical treatment, electroporation,microinjection, cell fusion, infection, vector mediated DNA transfer,bombardment, or Agrobacterium mediated transformation.

Preferred techniques are set forth below in Example 6 for transformationof maize plant cells and in Example 7 for transformation of soybeanplant cells.

Other preferred methods for transforming dicots, primarily by use ofAgrobacterium tumefaciens, and obtaining transgenic plants include thosepublished for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135,U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat.No. 5,416,011, McCabe et. al., Biol Technology 6:923 (1988), Christou etal., Plant Physiol. 87:671 674 (1988)); Brassica (U.S. Pat. No.5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653 657 (1996),McKently et al., Plant Cell Rep. 14:699 703 (1995)); papaya; and pea(Grant et al., Plant Cell Rep. 15:254 258, (1995)).

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have also been reported and are includedas preferred methods, for example, transformation and plant regenerationas achieved in asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA)84:5354, (1987)); barley (Wan and Lemaux, Plant Physiol 104:37 (1994));Zea mays (Rhodes et al., Science 240:204 (1988), Gordon-Kamm et al.,Plant Cell 2:603 618 (1990), Fromm et al., Biol Technology 8:833 (1990),Koziel et al., Biol Technology 11: 194, (1993), Armstrong et al., CropScience 35:550 557 (1995)); oat (Somers et al., Biol Technology 10: 1589 (1992)); orchard grass (Horn et al., Plant Cell Rep. 7:469 (1988));rice (Toriyama et al., Theor Appl. Genet. 205:34, (1986); Part et al.,Plant Mol. Biol. 32:1135 1148, (1996); Abedinia et al., Aust. J. PlantPhysiol. 24:133 141 (1997); Zhang and Wu, Theor. Appl. Genet. 76:835(1988); Zhang et al. Plant Cell Rep. 7:379, (1988); Battraw and Hall,Plant Sci. 86:191 202 (1992); Christou et al., Bio/Technology 9:957(1991)); rye (De la Pena et al., Nature 325:274 (1987)); sugarcane(Bower and Birch, Plant J. 2:409 (1992)); tall fescue (Wang et al., BiolTechnology 10:691 (1992)), and wheat (Vasil et al., Bio/Technology10:667 (1992); U.S. Pat. No. 5,631,152).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif.,(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells, culturing those individualizedcells through the usual stages of embryonic development through therooted plantlet stage. Transgenic embryos and seeds are similarlyregenerated. The resulting transgenic rooted shoots are thereafterplanted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous isolated nucleic acid fragment that encodes a protein ofinterest is well known in the art. Preferably, the regenerated plantsare self-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Atransgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

Assays to detect proteins may be performed by SDS-polyacrylamide gelelectrophoresis or immunological assays. Assays to detect levels ofsubstrates or products of enzymes may be performed using gaschromatography or liquid chromatography for separation and UV or visiblespectrometry or mass spectrometry for detection, or the like.Determining the levels of mRNA of the enzyme of interest may beaccomplished using northern-blotting or RT-PCR techniques. Once plantshave been regenerated, and progeny plants homozygous for the transgenehave been obtained, plants will have a stable phenotype that will beobserved in similar seeds in later generations.

The present invention also includes a method for determining whether aplant exhibits a ramosa3 mutant genotype comprising: (a) isolatinggenomic DNA from a subject; (b) performing a PCR on the isolated genomicDNA using the primer pair consisting of NS432 (SEQ ID NO:16) and NS411(SEQ ID NO:17); and (c) analyzing results of the PCR for the presence ofa larger DNA fragment as an indication that the subject exhibits theramosa3 mutant genotype.

Also included in the present invention is a method for determiningwhether a plant exhibits a ramosa3 mutant genotype comprising: (a)isolating genomic DNA from a subject; (b) performing a PCR on theisolated genomic DNA using any one of the following primer pairs: SEQ IDNOs:16 and 17; SEQ ID NOs:20 and 21; SEQ ID NOs:22 and 23; SEQ ID NOs:24and 25; SEQ ID NOs:26 and 27 or SEQ ID NOs:28 and 29; and (c) analyzingthe results of the PCR for the presence of a larger or smaller DNAfragment, relative to a non-mutant fragment, as an indication that thesubject exhibits the ramosa3 mutant genotype.

Another method included in the present invention is a method forselecting a first maize plant by marker assisted selection of aquantitative trait locus (“QTL”) associated with branching of thetassel, ear or both, the method comprising: determining the presence ofa locus in the first maize plant, wherein the locus hybridizes with afirst nucleic acid that is genetically linked to a nucleic acid sequencehaving at least 90% sequence identity, based on the Clustal V method ofalignment, when compared to SEQ ID NO:15; and selecting the first maizeplant comprising the locus that hybridizes with the first nucleic acid;thereby selecting the maize plant containing a QTL associated withbranching of the tassel, ear or both.

As described above, the present invention includes, among other things,compositions and methods for modulating (i.e., increasing or decreasing)the level of polypeptides of the present invention in plants. Inparticular, the polypeptides of the present invention can be expressedat developmental stages, in tissues, and/or in quantities which areuncharacteristic of non-recombinantly engineered plants. In addition toaltering (increasing or decreasing) branching, pollen shed ortrehalose-6-phosphate phosphatase activity, it is believed thatincreasing or decreasing the level of polypeptides of the presentinvention in plants also can also have an impact on yield by alteringthe numbers of fruits and seeds produced by the inflorescences (due toextra branches) or by making plants more compact allowing them to begrown under stringent conditions, e.g., planted at high density or underadverse weather conditions, such as drought. Thus, the present inventionalso provides utility in such exemplary applications as improvement ofyield or growth under stressful conditions.

The isolated nucleic acids and proteins and any embodiments of thepresent invention can be used over a broad range of plant types,particularly monocots such as the species of the Family Graminiaeincluding Sorghum bicolor and Zea mays. The isolated nucleic acid andproteins of the present invention can also be used in species from thegenera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago,Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Triticum, Bambusa,Dendrocalamus, and Melocanna.

EXAMPLES

The present invention is further illustrated in the following Examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Phenotypic Analysis of ra3 Mutants

A mutant line with branched inflorescences was isolated in anon-targeted Mutator (Mu) transposon tagging family, and found to be anallele of ramosa3 (ra3). Although ra3 is a classical mutant of maize,first described in 1954 by H. S. Perry (see also, Table 1 of Veit et al.Plant Cell 5:1205-1215 (1993)), it had not been characterized in detail.Only the mature inflorescence phenotype had been reported, and there wassome confusion about the map location, as explained below in Example 2.

To analyze ra3 mutant phenotypes, ra3 reference allele plants (ra3-ref;Maize Genetics CoOp Stock Center) were introgressed into B73 for atleast 3 generations. During vegetative development, no differences wereobserved in ra3 plants compared to wild type sibs. In the inflorescencestage, ra3 mutants had irregular long branches in the ears, whereas wildtype sib ears had a more compact and organized rachis. Also, irregularseed rows were present in contrast to the neatly organized rows inwild-type ears. See FIG. 1. As shown on FIG. 1, wild-type ear (A) hasnicely arranged rows and no long branches, whereas ra3 mutant ears (B)have long branches and irregular rows of kernels. Occasionally, ra3 earsare covered in long branches (B, right). Wild type tassels normally havelong branches at their base (C), and ra3 mutant tassels (D) have morebranches.

Mutant ra3 ears also had “upside-down” or inverted embryo orientations,which may indicate indeterminacy in the spikelet axis. These defects inra3 ears were usually seen only at the base, though occasionally theears were irregularly branched along their whole length (FIG. 1B). As inthe ear, ra3 tassels had more branches than wild-type tassels, althoughoverall the developmental defects appeared to be milder than in theears.

To determine how these defects in the mature inflorescences arise,scanning electron microscopy (SEM) was used to follow early ear andtassel development. The development of ears is described as follows.During the vegetative to inflorescence transition stage, there was nodifference between ra3 and wild-type (FIG. 2A, E), and the ra3inflorescence meristem (IM) initiated spikelet pair meristems (SPMs) asin wild-type (FIG. 2B, F). The first defects that were observed inimmature ra3 ears were enlarged and irregular sized SPMs, when the earprimordia were around 1.5 mm long. Slightly later in development, whenthe ear primordia were around 2 mm long, the conversion of SPMs toindeterminate meristems that produce additional SPMs was observed (FIG.2F, arrow 1). Because this SPM produced additional SPMs, we chose tocall it “branch meristem*” (BM*), to reflect its similarity to the BMsat the base of normal tassels. As ear development proceeded, the ra3SPMs made 3-4 glumes before the conversion to a BM* (FIG. 2G, arrows 2),while wild-type SMs made only 2 glumes that subtended the SM (arrows 2,FIG. 2C). Sometimes, floral meristems (FMs), rather than SMs, wereproduced inside these extra glumes in ra3 mutants (FIG. 2G). In summary,the SPMs that converted to BM* in ra3 ears had two possible identities,SPM* or a mixture of SPM and SM (SPM*+SM*). In this scheme, SPM*produced several glumes containing only SPMs and SMs, whereas “SPM*+SM*”produced several glumes subtending FMs.

Mutant ra3 SMs also showed other types of identity defects. For example,they often made multiple FMs (arrows 3, FIG. 2H), whereas wild-type SMsproduce only two FMs. Mutant ra3 SMs can even convert to BM* identityafter making several FMs. As in mature ears (FIG. 1), usually only themeristems in the lower half of the ear showed these defects.

In ra3 tassels, similar developmental defects as in ra3 ears wereobserved. For example, they also produced more indeterminate SPMs. Thisresulted in about double the number of long branches in ra3 tassels(wild-type 6.8±1.4; ra3 13.6±2.8 branches).

In summary, ra3 ears and tassels showed a range of phenotypic defects; asummary for the ear is presented in FIG. 3. The developmental analysisshowed that in general RA3 functions to impose determinacy and identityon the different meristem types in the inflorescences. Although the IMsare normal, the SPMs, SMs and FMs are all more indeterminate in ra3mutants compared to wild type.

Example 2 Mapping and Isolation of ra3

According to the maize genetic map available, in 2004, at the MaizeGenetics and Genomics Database, ra3 was listed as being mapped tochromosome 4; however, we were unable to reproduce those results. Toaddress this discrepancy, ra3 and sib DNAs were provided for analysis bythe Missouri Maize Mapping Project [Coe, E., Cone, K., McMullen, M.,Chen, S. S., Davis, G., Gardiner, J., Liscum, E., Polacco, M., Paterson,A., Sanchez-Villeda, H., Soderlund, C., and Wing, R., Access to themaize genome: an integrated physical and genetic map. Plant Physiol,2002. 128(1): p. 9-12].

Using bulk segregant analysis, ra3 was mapped to chromosome 7, bin 4.

For more detailed mapping studies, larger mapping populations wereconstructed using the ra3-ref allele and an allele (“ra3-feat”) that wasisolated from a Mu line carrying a fasciated ear mutant (feat, [Jackson,D. and Hake, S., The genetics of ear fasciation in maize. Maize GeneticsCooperation Newsletter, 1999. 73: p. 2]).

The ra3 and feat mutations segregated independently in this line,indicating that these mutations are in different genes. F2s of each ra3allele crossed to B73 were made, and high-resolution mapping wasconducted using simple sequence repeat (SSR), restriction fragmentlength polymorphism (RFLP), cleaved amplified polymorphic sequences(CAPS) and derived CAPS (dCAPS) markers (FIG. 4).

By analyzing 74 ra3-ref mutants in the F2, one recombinant betweenmarker csu597 (SEQ ID NOs:1 & 2) and ra3 and two recombinants indifferent individuals between marker umc1412 (SEQ ID NOs:3 & 4) and ra3were identified, placing the ra3 locus between these two markers. Tofurther delimit the ra3 region, a larger number of recombinants werescreened, and new CAPS markers, a14 (SEQ ID NOs:5 & 6) and n20 (SEQ IDNOs:7 & 8) were created using BAC end sequences covering part of the ra3region. DNA amplified from ra3-ref and B73 using these sets of primersgave polymorphisms when digested with Fok1 for a14 and HindIII for n20.Nine recombinants out of 873 ra3 mutants between al 4 and ra3 and 10recombinants between n20 and ra3 were obtained. The number of crossovers indicated that the genetic distance between ra3 and a14 was0.7+/−0.2 cM and the genetic distance between ra3 and n20 was 0.6+/−0.2cM.

This region of the maize genome was covered by three BAC clones,c0387K01, b0505C08 and b0063D15 [Cone, K. C. et al., Genetic, physical,and informatics resources for maize. On the road to an integrated map.Plant Physiol, 2002. 130(4): p. 1598-605]. New markers were made fromthese BACs by screening for non-repetitive DNA fragments. Southern blotswere made of each BAC clone digested with several restriction enzymes,and the blot was probed first with maize genomic DNA, and after imaging,probed again with the BAC DNA. Upon comparison of the two blot images,bands were identified that had a signal with the BAC hybridization butnot with total genomic DNA hybridization. These bands were cataloged asbeing non-repetitive, and designated as “cold bands” (cb, FIG. 4). Thesecold bands were used either as RFLP probes or, after sequencing, wereconverted into d-CAPS markers. The d-CAPS marker, cb.g1E, was made fromthe sequence of cold band e; the forward and reverse primers for cb.g1Eare given as SEQ ID NO:9 and SEQ ID NO:10, respectively. Using theseadditional markers, it was determined that the ra3 locus was positionedon the BAC c0387K01 (FIG. 4).

The nucleotide sequence of BAC c0387K01 was determined and this sequenceinformation was used to design primers for amplification of DNA from therecombinants identified earlier. NS346 (SEQ ID NO:11) and NS347 (SEQ IDNO:12) were one primer pair; NS362 (SEQ ID NO:13) and NS363 (SEQ IDNO:14) was a second primer pair. These two primer pairs were used togenerate PCR product length polymorphisms, which were used to delimitthe RA3 locus to a single predicted gene (FIG. 4).

Example 3 RA3 and SRA Gene Structure and Phylogenetic Analysis

The nucleotide sequence shown in SEQ ID NO:15 was deduced from BACc038K01, and encodes the RA3 gene with 3 kb of sequence upstream (5′) ofthe ATG initiation codon, and 7.5 kb downstream (3′) of the TGA stopcodon. Note that exons in SEQ ID NO:15 are displayed only for the regionfrom the start codon to the stop codon, and are not shown for the 5′ or3′ Untranslated Regions (5′- and 3′-UTRs).

The cDNA sequence corresponding to the RA3 region was isolated byreverse transcription of poly(A)-RNA followed by PCR. The QuiagenOneStep RT-PCR kit was used according to the manufacturersspecifications. The primer set used was NS432 (SEQ ID NO:16) and NS411(SEQ ID NO:17). SEQ ID NO:18 is the nucleotide sequence of theprotein-coding region deduced from the PCR product obtained using theNS432 and NS411 primers. The corresponding amino acid sequence of theRAMOSA3 polypeptide is shown as SEQ ID NO:19.

The RA3 gene encodes a predicted protein of 361 amino acids withsignificant similarity to trehalose-6-phosphate phosphatases (TPPs). Thepredicted polypeptide has a non-conserved N-terminal region of ˜80 aminoacids followed by the TPP domain which contains two “phosphatase boxes”(see FIG. 5, part A, region labeled “3”) [Goddijn, O. J. and van Dun,K., Trehalose metabolism in plants. Trends Plant Sci, 1999. 4(8): p.315-319; Thaller, M. C., Schippa, S., and Rossolini, G. M., Conservedsequence motifs among bacterial, eukaryotic, and archaeal phosphatasesthat define a new phosphohydrolase superfamily. Protein Sci, 1998. 7(7):p. 1647-52; Vogel, G., Aeschbacher, R. A., Muller, J., Boller, T., andWiemken, A., Trehalose-6-phosphate phosphatases from Arabidopsisthaliana: identification by functional complementation of the yeast tps2mutant. Plant J, 1998. 13(5): p. 673-83]. FIG. 5, part A contains an“A-domain” (SEQ ID NO:45) and a “B-domain” (SEQ ID NO:46), as designatedby Vogel et al. (1998) Plant J 13(5):673-83.

RA3 is very similar in the TPP domain to the functional TPPs, AtTPPA andAtTPPB, from Arabidopsis, and the A- and B-domains of RA3 and AtTPPB areidentical. In a comparison of plant TPP proteins to the correspondingyeast protein, RA3 is actually more similar to yeast TPS2 than is theArabidopsis AtTPPA (20% vs. 16% identity).

Adjacent to RA3 there was a highly similar TPP gene, which we havedesignated SISTER OF RAMOSA3 (SRA). The genomic DNA sequence containingthe SRA gene is shown in SEQ ID NO:47. The nucleotide sequence of theprotein-coding region of the SRA gene is shown in SEQ ID NO:48, and thecorresponding amino acid sequence of the SRA polypeptide is shown in SEQID NO:49. A cDNA clone, my.cs1.pk0072.d4, was prepared from RNA isolatedfrom the leaf and sheath of 5-week old Zea mays L. plants, and itcontains a fragment of the SRA gene. Part of the nucleotide sequence ofthe cDNA insert of clone my.cs1.pk0072.d4 is presented in SEQ ID NO:50.

In the region of conserved synteny in rice, only a single rice TPP gene,gi33146623 (SEQ ID NO:43), is found. RA3 and SRA have 60.4 and 59.3%overall sequence identity, respectively, with the rice TPP (Table 1).Genbank and the maize sequence assemblies available at the MaizeGenetics and Genomics Database and at The Institute for Genomic ResearchMaize Database were examined for the presence of closely relatedhomologs to RA3 and SRA. Because the maize proteins are not yetannotated we named them “ZmRA3 Like” (“ZmRA3L”).

A phylogenetic analysis of TPP genes from Arabidopsis, rice and maize isshown in a neighbor-joining tree (FIG. 5, part B). [Swofford, D., PAUP—ACOMPUTER-PROGRAM FOR PHYLOGENETIC INFERENCE USING MAXIMUM PARSIMONY.JOURNAL OF GENERAL PHYSIOLOGY, 1993. 102: p. A9] The tree indicates thatRA3, SRA and gi33146623 (SEQ ID NO:43) are most closely related, and RA3and SRA are likely paralogs. TPPB, a functional TPP from Arabidopsis(At1g78090; SEQ ID NO:53 corresponding to NCBI GI NO. 2944180) is themost closely related Arabidopsis protein.

FIGS. 6A-6D show a sequence alignment of the amino acid sequences forthe following trehalose-6-phosphate phosphatases: RA3 (SEQ ID NO:19);SRA (SEQ ID NO:49); rice TPP (SEQ ID NO:51); Arabidopsis TPPA (SEQ IDNO:52); Arabidopsis TPPB (SEQ ID NO:53); corn TPP (SEQ ID NO:54); andsoybean TPP (SEQ ID NO:55). Also shown are alignments with two truncatedforms of Arabidopsis TPPA and TPPB, in which the N-terminal 91 aminoacids have been removed from each. An asterisk above an amino acidresidue indicates that the position is totally conserved among the givenSEQ ID NOs, with respect to the Arabidopsis thaliana AtTPPB sequence.Below the sequences are shown two domains, A and B, that are conservedamong trehalose-6-phosphate phosphatases, as described in Vogel et al.(1998) Plant J 13(5):673-683. The given sequence for each conserveddomain is taken from the Arabidopsis thaliana AtTPPB amino acid sequenceat these positions. FIGS. 6A-6D indicate that regions of high sequencesimilarity are located in the carboxy-terminal 70% of the consensussequence. Vogel et al. have noted that the AtTPPA and AtTPPB proteinshave high sequence conservation to each other except for theamino-terminal 100 amino acids, which they note have features in commonwith chloroplast transit peptides. Vogel et al. have shown enzymeactivity for AtTPPA, AtTPPB, and a truncated AtTPPA polypeptide that ismissing the first 91 amino acids.

The data in Table 1 show the percent identity for each pair of aminoacid sequences from the group consisting of SEQ ID NOs:19, 49, 51, 52,53, 54, 55, an enzymatically active fragment of SEQ ID NO:52 (AtTPPA) inwhich the first 91 amino acids are missing (Vogel et al. (1998) Plant J13(5):673-683), and a corresponding truncated AtTPPB polypeptide inwhich the first 91 amino acids have been removed. From this comparisonof percent identities, the RA3 and SRA polypeptides were found to have ahigher percent identity to the AtTPPB truncated polypeptide, than theAtTPPA truncated polypeptide.

TABLE 1 Percent Sequence Identity of Amino Acid Sequences of PlantTrehalose-6-Phosphate Phosphatases With Each Other SEQ Percent Identityto SEQ ID NO: ID NO: 19 49 51 52 53 54 55 52t* 53t** 19 — 60.4 60.4 46.049.0 47.4 46.3 53.1 60.1 49 60.4 — 59.3 43.0 47.6 45.0 45.1 51.7 58.7 5160.4 59.3 — 44.5 48.1 47.5 45.1 53.4 59.4 52 46.0 43.0 44.5 — 46.5 55.360.2 100.0 56.9 53 49.0 47.6 48.1 46.5 — 48.0 46.0 54.8 100.0 54 47.445.0 47.5 55.3 48.0 — 54.2 60.5 59.4 55 46.3 45.1 45.1 60.2 46.0 54.2 —65.6 56.5 52t* 53.1 51.7 53.4 100.0 54.8 60.5 65.6 — 56.9 53t** 60.158.7 59.4 56.9 100.0 59.4 56.5 56.9 — *52t refers to a truncation of theAtTPPA polypeptide (SEQ ID NO: 52), in which 91 amino acids have beenremoved from the amino terminus. **53t refers to a truncation of theAtTPPB polypeptide (SEQ ID NO: 53), in which 91 amino acids have beenremoved from the amino terminus.

Example 4 Sequence Analysis of ra3 Mutant Alleles

To confirm that the predicted locus encodes RA3, seven ra3 alleles weresequenced. These ra3 alleles were either preexisting alleles or wereisolated from targeted screens. Each had a lesion in the candidate gene,indicating that it encodes RA3 (Table 2).

Coding regions of mutant alleles ra3-ref, ra3-feat, ra3-EV, ra3-NI,ra3-bre, ra3-JL and ra3-NS were sequenced following amplification usingthe following primer sets: 5′UTR-exon2 forward and reverse primers (SEQID NO:20 & 21, respectively); exon3-exon4 forward and reverse primers(SEQ ID NO:22 & 23, respectively); exon5-exon7 forward and reverseprimers (SEQ ID NO:24 & 25, respectively); exon8-exon10 forward andreverse primers (SEQ ID NO:26 & 27, respectively); and exon1′-3′UTRforward and reverse primers (SEQ ID NO:28 & 29, respectively).

The ra3-ref mutant allele has a 4 bp insertion in exon7 (SEQ ID NO:30).This results in a frame-shift after amino acid 249 and a premature stopcodon after 305 amino acids. The predicted ra3-ref polypeptide is shownin SEQ ID NO:31.

The ra3-fea1 mutant allele contains the following two mutations: 1) aninsertion of an ILS-1-like transposon element in the 5′-UTR region ofthe ra3-fea1 mutant gene (SEQ ID NO:32); and 2) a 4 bp insertion in thecoding sequence of exon7 (SEQ ID NO:33), leading to a frame-shift afteramino acid 258 and a premature stop codon after 305 amino acids. Thepredicted amino acid sequence of the ra3-fea1 polypeptide is shown inSEQ ID NO:34.

The ra3-EV mutant allele carries a 4 bp insertion in exon6 (SEQ IDNO:35). This results in a frame-shift after amino acid 224 and apremature stop codon after 305 amino acids. The predicted amino acidsequence of the ra3-EV polypeptide is shown in SEQ ID NO:36.

The ra3-NI mutant allele has an insertion of 141 by in exon10 (SEQ IDNO:37), leading to a different protein sequence after amino acid 333 anda premature stop codon after 335 amino acids. The predicted amino acidsequence of the ra3-NI polypeptide is shown in SEQ ID NO:38.

The ra3-bre mutant allele has a 10 bp insertion between exon6 and exon7(SEQ ID NO:39), leading to a frame-shift after amino acid 241 and apremature stop codon after 243 amino acids. The predicted amino acidsequence of the ra3-bre polypeptide is shown in SEQ ID NO:40.

The ra3-JL mutant allele has a deletion and rearrangement in exon6 (SEQID NO:41), leading to a different protein sequence after amino acid 217and a premature stop codon after 246 amino acids. The predicted aminoacid sequence of the ra3-JL polypeptide is shown in SEQ ID NO:42.

The ra3-NS mutant allele has a 2 bp insertion in exon6 (SEQ ID NO:43),leading to a frame-shift after amino acid 222 and a premature stop codonafter 299 amino acids. The predicted amino acid sequence of the ra3-NSpolypeptide is shown in SEQ ID NO:44.

The nature of some alleles was unusual, for example some allelesobtained from transposon screens had insertions of only a fewnucleotides, possibly reflecting abortive transposition events. Bothra3-ref and ra3-fea1 had a 4 bp insertion in exon 7, at differentpositions, and ra3-fea1 also contained a transposon in the 5′ region. Ineach allele the mutation caused a frame-shift, and a premature stopcodon.

TABLE 2 Seven ra3 Alleles Have Mutations in the Candidate Locus. AlleleSource Lesion ra3-ref Maize Genetics Stock 4 bp insertion into exon7Center ra3-fea1 Mu transposon stock (D. ~2 kb ILS-like transposonbetween Jackson) TATA box and exon1; 4 bp insertion into exon7 ra3-EVAc-Ds stock (E. 4 bp insertion into exon6 Vollbrecht) ra3-NI EMS screen(N. Inada) 141 bp insertion into exon10 ra3-bre Unknown (E. Irish) 10 bpinsertion in cDNA, between exon6 and exon7 ra3-JL Mu transposon screenDeletion and rearrangement in exon6 ra3-NS Spm transposon screen 2 bpinsertion into exon6

Each mutant allele has a stop codon before the second phosphatase box,except for ra3-NI, which has a stop codon after the second phosphatasebox. This correlates with phenotype, since ra3-NI mutants have themildest phenotype.

Example 5 RA3 Gene Expression Profile

RT-PCR analysis was used to determine where and when during developmentRA3 and SRA are expressed. First, RT-PCR primers were tested on the ra3alleles. In mRNA isolated from 1 cm long ear primordia, a transcript wasdetected in wild type (B73) ears and in ra3-EV, ra3-NI and ra3-bre.However, no transcript was detected in mRNA from ra3-feat immature ears,indicating the specificity of these primers for RA3 (FIG. 7A). Duringdevelopment, RA3 was expressed most strongly during early female andmale inflorescence development, and peaked at around 2-5 mm in ear andtassel development. At this stage, SPM and SM are being initiated on thedeveloping inflorescences. Very low levels of RA3 transcript weredetected in root or vegetative apex, and were not detected in leaf (FIG.7B). On the other hand, SRA was expressed more evenly throughoutdevelopment, with a slightly higher expression in root and larger tasselprimordia (FIG. 7B).

Additionally, in situ hybridization was used to determine if RA3expression was spatially regulated during early inflorescencedevelopment. RA3 expression was observed in ear primordia in acup-shaped group of cells at the base of SPMs, SMs and FMs, and at theboundary between upper and lower florets (FIG. 8). This expression isspecific for RA3, as it was not seen in ra3-ref ears. Together with thedevelopmental analysis, this highly restricted expression pattersuggests an important developmental role for RA3 in maize inflorescencedevelopment.

In summary, the RA3 gene of maize is expressed preferentially inrestricted domains at early stages of inflorescence development.Phenotypic analysis suggests that RA3 acts at this stage to restrict thedeterminacy and identity of different meristem types in theinflorescence.

Example 6 Prophetic Example Expression of Recombinant DNA in MonocotCells

A recombinant DNA construct comprising a cDNA encoding the instantpolypeptides in sense orientation with respect to the maize 27 kD zeinpromoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′end that is located 3′ to the cDNA fragment, can be constructed. ThecDNA fragment of this gene may be generated by polymerase chain reaction(PCR) of the cDNA clone using appropriate oligonucleotide primers.Cloning sites (NcoI or SmaI) can be incorporated into theoligonucleotides to provide proper orientation of the DNA fragment wheninserted into the digested vector pML103 as described below.Amplification is then performed in a standard PCR. The amplified DNA isthen digested with restriction enzymes NcoI and SmaI and fractionated onan agarose gel. The appropriate band can be isolated from the gel andcombined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. PlasmidpML103 has been deposited under the terms of the Budapest Treaty at ATCC(American Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209), and bears accession number ATCC 97366. The DNA segment frompML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kDzein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insertDNA can be ligated at 15° C. overnight, essentially as described(Maniatis). The ligated DNA may then be used to transform E. coliXL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterialtransformants can be screened by restriction enzyme digestion of plasmidDNA and limited nucleotide sequence analysis using the dideoxy chaintermination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical).The resulting plasmid construct would comprise a recombinant DNAconstruct encoding, in the 5′ to 3′ direction, the maize 27 kD zeinpromoter, a cDNA fragment encoding the instant polypeptides, and the 10kD zein 3′ region.

The recombinant DNA construct described above can then be introducedinto corn cells by the following procedure. Immature corn embryos can bedissected from developing caryopses derived from crosses of the inbredcorn lines H99 and LH132. The embryos are isolated 10 to 11 days afterpollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structuresproliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten 1 μg of plasmid DNAs are added to 50 μL ofa suspension of gold particles (60 mg per mL). Calcium chloride (50 μLof a 2.5 M solution) and spermidine free base (20 μL of a 1.0 Msolution) are added to the particles. The suspension is vortexed duringthe addition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains bialophos (5 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containingbialophos. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thebialophos supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 7 Prophetic Example Expression of Recombinant DNA in Dicot Cells

An expression cassette composed of the promoter from the B-conglycininor glycinin genes (Chen, Z-L, et al. (1988) EMBO J. 7:297-302), 5-primeto the cDNA fragment, can be constructed and be used for expression ofthe instant polypeptides in transformed soybean. The pinII terminatorcan be placed 3-prime to the cDNA fragment. Such construct may be usedto overexpress the instant polypeptides. It is realized that one skilledin the art could employ different promoters and/or 3-prime end sequencesto achieve comparable expression results.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos, which produce secondary embryos, arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromcauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 8 Prophetic Example Expression of Recombinant DNA in MicrobialCells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 isconstructed by first destroying the EcoRI and HindIII sites in pET-3a attheir original positions. An oligonucleotide adaptor containing EcoRIand Hind III sites is inserted at the BamHI site of pET-3a. This createspET-3aM with additional unique cloning sites for insertion of genes intothe expression vector. Then, the NdeI site at the position oftranslation initiation is converted to an NcoI site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, is converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% low melting agarose gel. Buffer and agarose contain 10μg/ml ethidium bromide for visualization of the DNA fragment. Thefragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°C. Cells are then harvested by centrifugation and re-suspended in 50 μLof 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 9 RAMOSA3 Protein—In Vitro Trehalose-6-Phosphate PhosphataseActivity

Protein expression and in vitro phosphatase assays. To producerecombinant proteins for in vitro phosphatase assays, E. coli expressionvectors were constructed containing polynucleotides encoding either theRA3 full-length protein, the RA3 TPP-domain fragment or the RA3N-terminal fragment. DNA fragments encoding either the RA3 full-lengthprotein, the RA3 TPP-domain fragment or the RA3 N-terminal fragment wereamplified, respectively, using the following primer pairs:

RA3 Full-Length Protein: NS487 (SEQ ID NO: 56): CGAGCCATGACGAAGCAC andNS429 (SEQ ID NO: 57): ATAAGCGCCTCTTTGCTGTTG; RA3 TPP-Domain Fragment:NS483 (SEQ ID NO: 58): GGCGACTGGATGGAGAAGCA and NS429 (SEQ ID NO: 57):ATAAGCGCCTCTTTGCTGTTG. RA3 N-Terminal Fragment: NS485 (SEQ ID NO: 59):GTGCGCGGATCCAGCCATGACGAAGCACGCCGCCTACTC and NS488 (SEQ ID NO: 60):CTTCTCGAATTCTCAGCCGTGCTCGGCGTCGGCG.PCR fragments were cloned into the vector pCR T7/NT-TOPO, whichintroduces an N-terminal histidine tag into the recombinant protein(Invitrogen). His-tagged recombinant proteins were expressed in E. coliand purified by a batch purification method (Qiagen). The proteins wereused at a concentration of 70 ng/μl for phosphatase assays using sugarphosphates (Sigma) at a concentration of 2 mM, or ser/thr phosphopeptideas described (Klutts, S. et al. “Purification, cloning, expression, andproperties of mycobacterial trehalose-phosphate phosphatase” J Biol Chem278:2093-2100 (2003)). Sugar phosphate phosphatase activity was measuredusing the following four sugar phosphates: trehalose-6-phosphate,glucose-6-phosphate, fructose-6-phosphate and sucrose-6-phosphate.Phosphate release was measured as OD₆₀₀ (serine/threonine phosphataseassay system, Promega).

His-tagged RA3 full-length protein (SEQ ID NO:61), His-tagged RA3TPP-domain fragment (SEQ ID NO:62) and the His-tagged Mycobacteriumtuberculosis trehalose-6-phosphate phosphatase (Edavana et al., ArchBiochem Biophys 426:250-257 (2004)) each catalyzed phosphate releasefrom trehalose-6-phosphate (T6P) but not from the other sugar phosphatesor the ser/thr phosphopeptide used as a reporter of protein phosphataseactivity (FIG. 9). The His-tagged RA3 N-terminal fragment (SEQ ID NO:63)had no phosphatase activity and the non-specific phosphatase, shrimpalkaline phosphatase (SAP; Roche Applied Science), showed phosphataseactivity against all substrates (FIG. 9). This in vitro activity datasupports the assignment of T6P phosphatase activity to the RA3 protein.

The His-tagged RA3 TPP-domain fragment (SEQ ID NO:62) consists of a35-aa N-terminal region that contains six consecutive histidineresidues, followed by amino acid residues 78-361 of the RA3 protein (SEQID NO:19). The in vitro activity data (FIG. 9) indicates that amino acidresidues 78-361 of the RA3 protein are sufficient to convey T6Pphosphatase activity.

Example 10 RAMOSA3 Protein—In Vivo

Trehalose-6-Phosphate Phosphatase Activity

Complementation of yeast tps2 mutant: To complement a yeast mutantdeficient in trehalose-6-phosphate phosphatase, DNA fragments encodingRA3 full-length protein and RA3 TPP-domain fragment each were clonedinto a yeast expression vector. DNA fragments encoding either the RA3full-length protein or the RA3 TPP-domain fragment were amplified,respectively, using the following primer pairs:

RA3 Full-Length Protein: NS489 (SEQ ID NO: 64):AAGGAAAAAAGCGGCCGCGCCATGACGAAGCACGCCGCCTACTC and NS490 (SEQ ID NO: 65):ACGAGGTCGTGCCTGCCGCTCATGGTTGGCGCGCCCCCTTCT; or RA3 TPP-Domain Fragment:NS490 (SEQ ID NO: 65): ACGAGGTCGTGCCTGCCGCTCATGGTTGGCGCGCCCCCTTCT andNS500 (SEQ ID NO: 66): CGCGCCGCCGGCGGCCGCGACATGGACTGGATGGAGAAGCACCCGTC.The DNA fragments encoding RA3 full-length protein and the RA3TPP-domain fragment were cloned into a yeast shuttle vector, pFL6, inwhich high-level expression is driven by the phosphoglycerate kinasepromoter (Minet, M., Dufour, M. E. & Lacroute, F. “Complementation ofSaccharomyces cerevisiae auxotrophic mutants by Arabidopsis thalianacDNAs” Plant J 2:417-422 (1992)). The Arabidopsis TPPB gene was used asa positive control. The empty yeast vector transformed into the yeastmutant served as a negative control. The constructs were transformedinto the yeast strain YSH6.106.-8C, which has a deletion of the tps2gene and hence lacks TPP activity. This mutant strain is sensitive tohigh temperature and salt concentrations (De Virgilio, C. et al.“Disruption of TPS2, the gene encoding the 100-kDa subunit of thetrehalose-6-phosphate synthase/phosphatase complex in Saccharomycescerevisiae, causes accumulation of trehalose-6-phosphate and loss oftrehalose-6-phosphate phosphatase activity” Eur. J. Biochem 212:315-323(1993)). The yeast TPP mutant grows normally at 30° C., but has veryslow growth at elevated temperature (40° C.), especially in the presenceof an osmotic stress such as 1M NaCl. Transformed cells were assayed forgrowth on selective media at 40.5° C., the non-permissive temperature,in the presence of 1M NaCl, as well as at 30° C.

RA3 full-length protein (SEQ ID NO:19) and RA3 TPP-domain fragment (SEQID NO:68) each rescued growth at the non-permissive temperature (FIG.10). Consequently, RA3 protein functions as a T6P phosphatase in vivo.

The RA3 TPP-domain fragment (SEQ ID NO:68) expressed in the yeast mutantconsists of a start methionine residue followed by amino acid residues79-361 of the RA3 protein (SEQ ID NO:19). The data in FIG. 10 indicatesthat amino acid residues 79-361 of the RA3 protein are sufficient toconvey T6P phosphatase activity in vivo.

1. (canceled)
 2. (canceled)
 3. An isolated polynucleotide comprising:(a) a nucleic acid sequence encoding a polypeptide havingtrehalose-6-phosphate phosphatase activity, wherein the polypeptide hasan amino acid sequence comprising SEQ ID NO:69; or (b) a complement ofthe nucleotide sequence, wherein the complement and the nucleotidesequence consist of the same number of nucleotides and are 100%complementary.
 4. (canceled)
 5. (canceled)
 6. A recombinant DNAconstruct comprising the polynucleotide of claim 3 operably linked to apromoter that is functional in a plant. 7-12. (canceled)
 13. A methodfor altering trehalose-6-phosphate phosphatase activity in a plant,comprising: (a) introducing into a regenerable plant cell therecombinant DNA construct of claim 6 to produce a transformed plantcell; and (b) regenerating a transgenic plant from said transformedplant cell, wherein said transgenic plant comprises in its genome saidrecombinant DNA construct and wherein said transgenic plant exhibits analteration in trehalose-6-phosphate phosphatase activity, when comparedto a control plant not comprising said recombinant DNA construct. 14.The method of claim 13, further comprising (c) obtaining a progeny plantderived from said transgenic plant, wherein said progeny plant comprisesin its genome the recombinant DNA construct.
 15. The method of claim 13,wherein the transgenic plant exhibits an increase intrehalose-6-phosphate phosphatase activity.
 16. A method for increasingenvironmental stress tolerance of a plant, comprising: (a) introducinginto a regenerable plant cell the recombinant DNA construct of claim 6to produce a transformed plant cell; and (b) regenerating a transgenicplant from said transformed plant cell, wherein said transgenic plantcomprises in its genome said recombinant DNA construct and wherein saidtransgenic plant exhibits an increase in environmental stress tolerance,when compared to a control plant not comprising said recombinant DNAconstruct.
 17. The method of claim 16, further comprising (c) obtaininga progeny plant derived from said transgenic plant, wherein said progenyplant comprises in its genome the recombinant DNA construct.
 18. Themethod of claim 16, wherein said environmental stress is drought, andwherein said transgenic plant exhibits an increase in drought tolerance.